14-3-3zeta/leonardo

REGULATION

Protein Interactions

Slob has been identified as a novel protein that binds to the carboxy-terminal domain of Slowpoke. A yeast two-hybrid screen with Slob as bait identifies the zeta isoform of 14-3-3 as a Slob-binding protein. All three proteins are colocalized presynaptically at Drosophila neuromuscular junctions. 14-3-3 is known to be highly enriched in synaptic boutons at the neuromuscular junction and is present only at much lower levels in the motor axon and muscle (Broadie, 1997). Slob is also enriched in synaptic boutons, although its distribution appears to be less restricted than that of 14-3-3. Both 14-3-3 and dSlo are prominent in synaptic boutons, where they colocalize. Two serine residues in Slob are required for 14-3-3 binding, and the binding is dynamically regulated in Drosophila by calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation of these residues. Slob itself increases the voltage sensitivity of dSlo, whereas 14-3-3 decreases the channel's voltage sensitivity (Zhou, 1999).

What are the molecular details of the profound downregulation of dSlo channel activity by 14-3-3? Members of the family of K_Ca channels are subject to modulation by a variety of molecular mechanisms, ranging from protein phosphorylation to oxidation/reduction reactions. It is conceivable that simply the binding of 14-3-3 to dSlo via Slob is sufficient to alter the gating of the channel, as appears to be the case for ß subunit interactions with Slowpoke and other potassium channels. Alternatively, 14-3-3 may act as another scaffolding component, to bring one of the protein kinases that it is known to bind into the proximity of the channel. Indeed, because 14-3-3 dimerizes, it might bridge the interactions of several different signaling proteins with the channel. It will be interesting to determine whether the Raf protein kinase, one of the kinases that binds 14-3-3, can phosphorylate and modulate dSlo, because Raf is a key player in the mitogen-activated protein (MAP) kinase pathway that conveys signals from the plasma membrane to the cell nucleus. Activation of this pathway can influence ion channel expression and activity, and potassium channel activity in turn can modulate tyrosine kinase signaling in cells. Thus, the present findings raise the intriguing possibility that a potassium channel regulatory complex is involved in MAP kinase signaling and the regulation of many fundamental cell processes (Zhou, 1999 and references).

The finding that the interaction of Slob with 14-3-3 requires Slob phosphorylation is consistent with studies of other 14-3-3 binding proteins. It is especially intriguing that the binding can be regulated in vivo by changes in the activity of CaMKII; these results suggest that there may be dynamic physiological regulation of dSlo channel activity by 14-3-3 that depends on the phosphorylation state of Slob. In view of the presynaptic colocalization of the three proteins described here, it is interesting that it is the CaMKII phosphorylation of Slob that regulates 14-3-3 binding. CaMKII is also present at a high concentration presynaptically in Drosophila, and thus the same calcium rise that evokes transmitter release might promote phosphorylation of Slob, binding of 14-3-3, and downregulation of dSlo (Zhou, 1999 and references).

14-3-3 eta is required for photoreceptor development in Drosophila, while another isoform, 14-3-3 epsilon, also influences photoreceptor development by regulating Ras-mediated signaling pathways. It is particularly interesting that flies lacking 14-3-3 zeta are severely impaired in an olfactory learning task and exhibit defects in basal synaptic transmission as well as in synaptic plasticity (Broadie, 1997). 14-3-3 zeta is enriched in presynaptic boutons of the neuromuscular junction (Broadie, 1997), consistent with a role in synaptic transmission. This presynaptic localization of 14-3-3 zeta is confirmed and in addition it colocalizes with both dSlo and Slob in presynaptic boutons. Slowpoke channels are also enriched in presynaptic endings in rat brain and frog neuromuscular junction, where they influence transmitter release. Since dSlo current contributes to membrane repolarization and helps to limit transmitter release from Drosophila nerve terminals, and 14-3-3 downregulates dSlo via Slob, it is plausible that there is greater nerve terminal dSlo current in 14-3-3 mutant flies and that this accounts for the decreased synaptic transmission seen in these mutants (Broadie, 1997). The possibility that a modulatory complex associated with a neuronal ion channel may influence synaptic transmission, and ultimately higher brain functions, is an attractive hypothesis for future investigation (Zhou, 1999 and references).

14-3-3 proteins mediate PAR-1 function in axis formation

PAR-1 kinases are required to determine the anterior-posterior (A-P) axis in C. elegans and Drosophila, but little is known about their molecular function. Drosophila 14-3-3 proteins, 14-3-3epsilon and 14-3-3zeta/Leonardo (Leo) represent the Drosophila homologs of C. elegans PAR-5. 14-3-3 proteins have been identified as Drosophila PAR-1 interactors; PAR-1 binds a domain of 14-3-3 distinct from the phosphoserine binding pocket. PAR-1 kinases phosphorylate proteins to generate 14-3-3 binding sites and may therefore directly deliver 14-3-3 to these targets. 14-3-3 mutants display phenotypes identical to par-1 mutants in oocyte determination and the polarization of the A-P axis. Together, these results indicate that PAR-1's function is mediated by the binding of 14-3-3 to its substrates. The C. elegans 14-3-3 protein, PAR-5, is also required for A-P polarization, suggesting that this is a conserved mechanism by which PAR-1 establishes cellular asymmetries (Benton, 2002).

PAR-1 contains three conserved domains: centrally-located kinase and ubiquitin-associated (UBA) domains, and a C-terminal domain of unknown function. Since the C-terminal domain is dispensable for PAR-1 function in the germline, a yeast two-hybrid screen was performed using a bait containing the kinase and UBA domains. The largest class of preys, representing over 25% of the recovered clones, corresponded to the two Drosophila 14-3-3 proteins, 14-3-3epsilon and 14-3-3zeta/Leonardo (Leo). These interactors represent the Drosophila homologs of C. elegans PAR-5, and this interaction appears to be conserved, since PAR-5 can bind to a fragment of C. elegans PAR-1 (Benton, 2002).

To confirm this interaction by an independent assay, in vitro-synthesized, labeled full-length Drosophila PAR-1 was incubated with bacterially expressed maltose binding protein (MBP)-tagged 14-3-3 proteins bound to amylose beads. Beads containing MBP:14-3-3 fusion proteins, but not MBP alone, efficiently precipitate PAR-1, indicating that this interaction is direct (Benton, 2002).

14-3-3 proteins regulate the activity or subcellular localization of a diverse set of proteins, including several protein kinases, by binding in a phosphorylation-dependent manner to conserved motifs (RSXpSXP or RX_1-2pSX_2-3pS). Using the yeast two-hybrid system, it was found that 14-3-3 appears to associate with the kinase domain of PAR-1. This contrasts with the interaction of 14-3-3 with other kinases, such as Raf and Wee1, in which 14-3-3 recognizes a phosphoserine-containing motif lying outside the catalytic domain. This interaction with PAR-1 is kinase specific, since 14-3-3 does not bind to the catalytic domains of PKA or aPKC (Benton, 2002).

The region of 14-3-3epsilon that interacts with PAR-1 was determined using the molecular information of three missense alleles of 14-3-3epsilon (Chang, 1997). These alleles were isolated as suppressors of activated Ras or Raf and impair the function of 14-3-3 in Ras/Raf/MAPK signaling. One mutation, E183K, lies within the phosphoserine binding pocket and affects a residue that directly contacts phosphoserine-peptide ligands. The others, F199Y and Y214F, are both located outside this pocket in a hydrophobic region of unknown function. Each of these three mutations were introduced into a 14-3-3epsilon prey clone and their effects on the intermolecular interactions of 14-3-3epsilon were tested (Benton, 2002).

Since 14-3-3 proteins function as dimers, whether these mutations influenced the dimerization property of 14-3-3epsilon was tested. 14-3-3epsilon can form both homodimers and heterodimers with Leo, and none of the three mutations significantly affects these interactions. This is consistent with the location of these mutations in regions distinct from the dimerization interface and indicates that global protein structure and stability are not affected. Interactions were tested of these mutant proteins with a domain of Drosophila Raf that contains a conserved 14-3-3-recognition motif (R⁷⁴⁰SApSEP⁷⁴⁵). Raf binds to both Drosophila 14-3-3 isoforms, and the interaction with 14-3-3epsilon is completely abolished by the E183K mutation, but not by the F199Y and Y214F mutations, as expected for an association via the phosphoserine binding pocket (Benton, 2002).

These mutations have opposite effects upon the interaction with PAR-1: E183K does not impair binding, whereas the other mutations either result in a severe (F199Y) or a more modest (Y214F) reduction in the strength of this interaction. These results indicate that 14-3-3epsilon does not bind PAR-1 via its phosphoserine binding pocket, consistent with the lack of canonical binding motifs in PAR-1. The interaction instead appears to be mediated by a novel interface on the external surface of the 14-3-3 molecule. Since the F199 and Y214 residues are conserved in 99% of 14-3-3 sequences, this interface is likely to exist in all isoforms (Benton, 2002).

Since binding of PAR-1 to 14-3-3 should leave the phosphoserine binding pocket vacant, and PAR-1 is a serine/threonine kinase, it was reasoned that PAR-1 might be involved in regulating the phosphorylation-dependent interactions of 14-3-3 with other proteins. Whether PAR-1 can phosphorylate proteins to generate the phosphoserine epitope recognized by 14-3-3 was tested. Using either immunoprecipitated or bacterially expressed PAR-1, efficient phosphorylation was observed of the 14-3-3-interacting portion of Raf. Phosphorylation of Ser743, or the equivalent residue in Raf homologs, is essential for 14-3-3 binding and for Raf function in vivo. This residue was mutated to alanine and this was found to completely abolish the phosphorylation by PAR-1, indicating that PAR-1 specifically phosphorylates Raf to generate a 14-3-3 binding site. This activity of PAR-1 does not require the presence of 14-3-3, and addition of 14-3-3 to this assay does not detectably affect Raf phosphorylation. A mammalian PAR-1 homolog, C-TAK1, is able to phosphorylate proteins such as KSR and Cdc25C within 14-3-3 binding sites, suggesting that this specificity is a conserved property of this kinase family (Benton, 2002).

To test whether 14-3-3 proteins are involved in par-1-dependent processes in vivo, loss-of-function mutations in 14-3-3epsilon and leo were analyzed. Surprisingly, flies homozygous for a protein null allele of 14-3-3epsilon (14-3-3epsilon^j2B10) are viable. However, females lay very few eggs, which fail to hatch. Most egg chambers from these females lack differentiated oocytes, as revealed by DNA staining, which distinguishes the oocyte karyosome from the 15 polyploid nurse cells. An identical phenotype is observed in ovaries from flies containing this allele over a deficiency, indicating that the phenotype is specific for this locus. To determine where 14-3-3epsilon is required, clones of this allele were generated in either the germline or somatic follicle cells. Defects in oocyte differentiation were observed only in germline clones; thus, like PAR-1, 14-3-3epsilon is required in the germline for oocyte differentiation (Benton, 2002).

Oocyte determination depends on the MT-dependent transport of specific factors, such as Orb and the germ cell centrosomes, to one cell in the cyst. These factors initially concentrate at the anterior of this cell but subsequently translocate around the nucleus and concentrate along the posterior cortex. This second step appears to require the establishment of a diffuse MTOC along the posterior of the cell and is essential for its stable determination as the oocyte. The formation of this MTOC can be visualized using an antibody to Minispindles (MSPS), a MAP that localizes to sites of MT nucleation. In wild-type egg chambers, MSPS accumulates along the posterior cortex. This accumulation is undetectable in 14-3-3epsilon mutants, indicating that the MTOC has failed to form. Orb and the centrosomes therefore do not undergo the anterior-to-posterior movement and eventually diffuse away as this cell exits meiosis and adopts a nurse cell fate. These phenotypes are indistinguishable from those of par-1 null mutant cysts, indicating that 14-3-3epsilon and PAR-1 function together in this specific step of oocyte determination (Benton, 2002).

In contrast to 14-3-3epsilon mutants, germline clones of a strong lethal allele of leo (leo^P1188) display no defects in this process. Since the 14-3-3epsilon phenotype is incompletely penetrant, whether 14-3-3 proteins have partially redundant functions in the germline was tested. Removal of one copy of leo in 14-3-3epsilon mutant clones results in a fully penetrant defect in oocyte determination. Furthermore, removal of one copy of 14-3-3epsilon in leo mutant cysts uncovers an important contribution of leo in this process, since 84% of these cysts display defects in Orb localization. Thus, although 14-3-3epsilon has the predominant function in oocyte determination, Leo can partially compensate in its absence (Benton, 2002).

Polyclonal antibodies against 14-3-3epsilon and Leo were used to examine their localization in the germline. These antibodies are specific, since they do not stain tissue mutant for the corresponding isoform. 14-3-3epsilon is highly expressed in the dividing germline cells in the germarium, and colocalizes with PAR-1 on the fusome, a membranous structure that branches into each germ cell during the early germ cell divisions. The asymmetric partitioning of the fusome during these divisions results in one cell always inheriting more fusome material, which may provide an initial cue to specify this cell as the oocyte. The colocalization of PAR-1 and 14-3-3epsilon on the fusome may therefore represent a mechanism to concentrate these proteins in the future oocyte (Benton, 2002).

At later stages, 14-3-3epsilon colocalizes with PAR-1 at the ring canals, which interconnect the germline cells in each cyst. 14-3-3epsilon can be detected in the cytoplasm and around the cortex of the oocyte but, unlike PAR-1, does not accumulate at the posterior pole. Leo is also expressed in the germline and displays a similar localization to ring canals, but is expressed at very low levels in the germarium (Benton, 2002).

While the fusome and ring canals may represent sites of physical and functional association of PAR-1 with 14-3-3, its localization to these sites is not affected in 14-3-3 mutants, indicating that 14-3-3 binding does not simply act to target PAR-1 to these subcellular destinations. Mutations in the Drosophila PAR-3 homolog Baz cause similar phenotypes in oocyte determination as par-1 and 14-3-3 mutants. However, Baz concentrates at distinct sites in the germarium, in circles around each ring canal that also contain components of adherens junctions, and this localization is not detectably affected in 14-3-3 mutants (Benton, 2002).

The high early cytoplasmic concentration of 14-3-3epsilon has prevented an conclusive determination of whether the fusome localization of 14-3-3epsilon is PAR-1 dependent. However, 14-3-3epsilon is detectable at ring canals in cysts homozygous for a par-1 null allele (Benton, 2002).

To determine if 14-3-3 proteins function with PAR-1 in the repolarization of the oocyte to define the A-P axis, the distribution of osk mRNA and Stau was examined in late-stage egg chambers recovered from homozygous and hemizygous 14-3-3epsilon^j2B10 females, and in germline clones of this allele. These mutants display a partially penetrant phenotype, in which osk mRNA and Stau accumulate in dots in the middle of the oocyte. Twenty seven percent of egg chambers display both ectopic and posterior accumulation of osk mRNA and Stau protein, and four percent contain only mislocalized dots. These defects are very similar to those of hypomorphic par-1 mutants and can be strongly enhanced by removal of one copy of par-1 (Benton, 2002).

Although most bcd mRNA localizes normally to the anterior cortex in 14-3-3epsilon mutants, a small proportion is mislocalized along the lateral cortex, and occasionally at the posterior. In contrast to previous observations, such defects in bcd mRNA distribution are also observed in par-1 mutants. These are more pronounced at stages 8-9 than at stage 10, which might reflect a partial recovery in bcd mRNA localization to the anterior between these stages or the diffusion of the mRNA away from the lateral and posterior cortices due to a failure in anchoring. Other mutants that affect the localization of bcd and osk mRNAs, such as gurken, also disrupt the migration of the oocyte nucleus to the dorsal-anterior corner. As in par-1 mutants, however, oocyte nucleus migration appears to be unaffected in 14-3-3epsilon^j2B10 mutants (Benton, 2002).

Oocytes that are homozygous for leo^P1188 do not display polarity defects. However, strong dominant genetic interactions are observed between 14-3-3 mutants. Thus, these isoforms also function partially redundantly in this process (Benton, 2002).

To gain insights into the basis for the defects in mRNA localization, the organization of the MT cytoskeleton was examined, using a MT plus end marker, Kin:ß-gal. This marker accumulates at the posterior pole in wild-type oocytes, suggesting that the majority of MT plus ends are focused on this site. In contrast, Kin:ß-gal concentrates in the center of 14-3-3epsilon mutant oocytes, indicating that MT plus ends are focused incorrectly, and providing an explanation for the defects in osk mRNA/Stau distribution. The organization of oocyte MTs was directly analyzed using both a FITC-conjugated anti-alpha-tubulin antibody and a Tau:GFP reporter of MT distribution in living egg chambers . In contrast to the wild-type anterior-to-posterior gradient of MTs, 14-3-3epsilon mutants show a uniform distribution of MTs around the oocyte cortex, with the lowest density of MTs in the center. These defects in MT organization are indistinguishable from those of par-1 mutants (Benton, 2002).

The combination of phenotypes in osk and bcd mRNA localization and MT organization is, thus far, unique to par-1 and 14-3-3 mutants, and strongly suggests that they function together in the polarization of the A-P axis (Benton, 2002).

To determine the importance of the 14-3-3 protein interaction domains in vivo, the phenotypes of the 14-3-3epsilon missense alleles, 14-3-3epsilon^F199Y and 14-3-3epsilon^E183K, were characterized. Neither mutation significantly affects the level or localization of the protein, as assessed by immunostainings. 14-3-3epsilon^E183K displays penetrant defects in both oocyte determination and polarization. The penetrance of the latter is almost three times that observed with the protein null allele, indicating that the E183K mutant protein functions as a dominant negative, presumably through the formation of nonfunctional heterodimers with Leo. Thus, the interaction of 14-3-3 dimers with phosphorylated targets is critical for its function in the germline (Benton, 2002).

14-3-3epsilon^F199Y mutant egg chambers do not exhibit significant defects in oocyte determination or polarization, consistent with previous reports that this allele only displays phenotypes under genetically sensitized conditions (Chang, 1997). In the absence of leo, however, this allele has a dominant phenotype, with 11% of leo^P1188;14-3-3epsilon^F199Y/+ egg chambers displaying defects in oocyte determination. Thus, the PAR-1 interaction interface is also important for 14-3-3 function (Benton, 2002).

Thus loss-of-function mutations in 14-3-3 cause phenotypes identical to par-1 mutants in both the initial polarization of the oocyte and the repolarization that defines the A-P axis. These results indicate that 14-3-3 functions as an essential cofactor for PAR-1 in the generation of polarity (Benton, 2002).

Given the diverse roles of 14-3-3 proteins, it is very surprising that the only essential requirement for 14-3-3epsilon is in PAR-1-dependent polarization events in the Drosophila germline. A similar dedication of 14-3-3 function may exist in C. elegans, where animals homozygous for hypomorphic mutations in the 14-3-3 isoform encoded by par-5 are viable but give rise to progeny with highly penetrant defects in the polarization of the A-P axis. Indeed, the discovery that 14-3-3 is required for the initial polarization of the oocyte in the germarium reveals a remarkable homology between the generation of the first A-P asymmetries in flies and worms. Mutations in 14-3-3epsilon give a very specific defect in oocyte determination, in which the oocyte is initially specified correctly but fails to establish a posterior MTOC and to translocate oocyte-specific factors from the anterior to the posterior cortex. This phenotype is identical to that of par-1 null mutants, and the colocalization of PAR-1 and 14-3-3 on the fusome supports the idea that they function together in this process (Benton, 2002).

The Baz/PAR-6/aPKC complex is also required for this step of oocyte determination but localizes to a distinct site in the germarium. Furthermore, it has recently been shown that mutants in the Drosophila homolog of PAR-4 display this phenotype (S. Martin and D.S.J., unpublished data reported in Benton, 2002). Thus, this early polarization of the oocyte requires the Drosophila homologs of five of the six par genes that mediate the A-P polarization of the C. elegans zygote. The final gene, par-2, has no obvious homologs in other organisms and may perform some function that is unique to C. elegans (Benton, 2002).

Although the full complement of PAR proteins is necessary for the initial polarization of the Drosophila oocyte in the germarium, the Baz/PAR-6/aPKC complex does not appear to be required for the repolarization of the oocyte at stage 7. In baz and par-6 null germline clones, a few egg chambers escape the block in oocyte determination, and these complete oogenesis normally, displaying no defects in the localization of Stau to the posterior. Thus, the PAR-1/14-3-3 complex can function to polarize the oocyte independently of these other PAR proteins. PAR-1 also is required for the apical-basal polarity of the follicular epithelium, and localizes to the basolateral domain in these cells. It is interesting to note that 14-3-3epsilon concentrates basolaterally in follicle cells, raising the possibility that it functions with PAR-1 in this process as well. The PAR-1/14-3-3 complex may therefore represent a conserved polarity 'cassette' that plays an analogous role to the Baz/PAR-6/aPKC complex. This requirement is not universal, however, because PAR-1 does not appear to be necessary for the apical-basal polarization of the neuroblasts (J. Kaltschmidt and R. B., unpublished data reported in Benton, 2002), which depends upon Baz, PAR-6, and aPKC. Thus, the two PAR protein complexes may comprise distinct modules that can function either together or separately to generate polarity in different contexts (Benton, 2002 and references therein).

While the common requirement for the PAR proteins strongly suggests that the mechanisms that generate the first A-P asymmetries are conserved between flies and worms, the regulatory relationships between these proteins are not conserved. The hierarchy of PAR protein function in C. elegans has been inferred from the effects of mutants in each par gene on the localization of the other PAR proteins. This analysis places PAR-5 at the top of the hierarchy because it is required for the anterior localization of the PAR-3/PAR-6/PKC-3 complex and the posterior localization of PAR-2 and PAR-1, whereas PAR-1 lies at the bottom because par-1 mutants have little effect on the asymmetric localization of other PAR proteins. In contrast, in Drosophila, Baz and PAR-1 are localized normally in 14-3-3 mutants. Furthermore, although the localization of members of the PAR-3/PAR-6/PKC-3 complex are codependent in the C. elegans zygote, this is not the case in the Drosophila oocyte, nor are they required for the localization of PAR-1 to the fusome. The different positions of PAR-5 and PAR-1 in the C. elegans hierarchy indicate that PAR-5 functions independently of PAR-1 in the localization of the other PAR proteins, but this early requirement makes it difficult to assess whether it is also necessary at other stages in the pathway. The results in Drosophila and the observation that C. elegans PAR-5 and PAR-1 interact in yeast raise the possibility that PAR-5 also functions downstream of PAR-1 (Benton, 2002).

Although the results indicate that PAR-1 and 14-3-3 function together to polarize the oocyte at two stages of oogenesis, the mechanisms by which they generate these polarities are unknown. The repolarization of the oocyte at stage 7 principally affects the organization of the MTs. The original posterior MTOC is disassembled, and the MTs are reorganized to form an A-P gradient, in which most MTs appear to be nucleated from the anterior cortex, with their plus ends extending toward the posterior pole. In 14-3-3 and par-1 mutants the MTs are evenly distributed around the cortex, and a MT plus end marker and osk mRNA/Stau localize to the center of the oocyte. These observations led to the proposal that PAR-1 functions to recruit the plus ends to the posterior. This study shows that par-1 and 14-3-3 mutants also display mislocalization of bcd mRNA around the cortex. Since this mRNA is believed to be transported to the minus ends of MTs, this suggests that MTs are abnormally nucleated from all regions of the oocyte. Thus, PAR-1 and 14-3-3 may also contribute to the generation of the MT gradient by specifically inhibiting MT nucleation along the posterior and lateral cortices. The role of PAR-1 and 14-3-3 in the initial polarization of the oocyte in the germarium is also likely to involve MTs since their loss results in a failure in the formation of an MTOC at the posterior of the cell. The mechanisms that control the formation of this MTOC are not known, however, and it is unclear whether PAR-1 and 14-3-3 function in the same way to polarize the oocyte at both stages (Benton, 2002).

A model for 14-3-3 function with PAR-1 is presented. 14-3-3 proteins regulate the activity of numerous cellular proteins in a phosphorylation-dependent manner by binding as dimers to phosphoserine/threonine-containing motifs. In many cases, this regulation involves sequestration of the target protein in the cytoplasm. For example, 14-3-3 binding to the proapoptotic factor Bad blocks its translocation to mitochondria. 14-3-3 can also directly regulate the activity of its targets: the association of 14-3-3 with serotonin N-acetyltransferase, for example, enhances its ability to bind substrates. The interaction of 14-3-3 with PAR-1 differs from these canonical 14-3-3/target interactions in several respects: (1) the binding of 14-3-3 does not appear to regulate PAR-1 activity, since 14-3-3 mutants have no effect on PAR-1 localization or stability in vivo, or on kinase activity in vitro; (2) the PAR-1 kinase domain lacks both of the well-defined 14-3-3 binding motifs, and interacts with a novel hydrophobic region that is distinct from the phosphoserine binding pocket, which should therefore still be available to bind to other proteins. Thus, 14-3-3 may act as a cofactor for PAR-1 by binding to proteins that are phosphorylated by the kinase. In support of this, it has been demonstrated that PAR-1 can specifically phosphorylate a 14-3-3 binding site in Raf (Benton, 2002).

These observations suggest a model in which PAR-1 has a dual role in regulating 14-3-3/target interactions, first by generating the 14-3-3 binding phosphoepitope, and second by directly delivering 14-3-3 to these sites. Once 14-3-3 is bound to target proteins, its continued association with PAR-1 would maintain the kinase in close proximity to its substrate, which might ensure the stable maintenance of the phosphorylated state (Benton, 2002).

In addition to their role in establishing cell polarity, PAR-1 kinases have been implicated in a diverse range of other cellular processes. The closest mammalian homolog of PAR-1, C-TAK1, was initially purified as an activity that phosphorylates Cdc25C on Ser216. The in vivo significance of this regulation is unknown, but phosphorylation of this site by a distinct kinase, Chk1, induces 14-3-3 binding, and this inhibits Cdc25C as part of the DNA damage checkpoint. C-TAK1 also phosphorylates KSR to promote 14-3-3 binding, which sequesters KSR in the cytoplasm and inhibits EGF signaling. These biochemical activities of C-TAK1 are consistent with the data in Drosophila showing that PAR-1 phosphorylates a 14-3-3 binding site in Raf, and that 14-3-3 mutants give identical phenotypes to par-1 mutants in the germline. The ability to phosphorylate 14-3-3 binding sites may be a general property of PAR-1 kinases, which accounts for the diversity of their functions (Benton, 2002).

Consistent with this, other PAR-1 substrates have been shown to associate with 14-3-3 or contain conserved potential 14-3-3 recognition motifs. The vertebrate PAR-1 homologs, MARK1 and MARK2, were identified as kinases that phosphorylate Tau to inhibit its MT binding ability. 14-3-3 interacts with the MT binding domain of Tau and appears to compete with tubulin for Tau binding. MARK kinase regulation of Tau may therefore be mediated through 14-3-3, which physically blocks the association of Tau with MTs. PAR-1 also phosphorylates the Wingless pathway component Dishevelled. This phosphorylation has been mapped to a 30 amino acid region of the protein, which contains a putative 14-3-3 recognition motif (amino acids 234-242: RTSSYSS) that is essential for its function in planar polarity (Benton, 2002).

The intimate functional relationship between PAR-1 and 14-3-3 raises the possibility that this kinase might be involved in regulating other processes involving 14-3-3 proteins. For example, the observation that PAR-1 phosphorylates Raf to generate a 14-3-3 binding site makes it a candidate for the unidentified kinase that regulates Raf in vivo. In support of this, this study shows that the F199Y and Y214F mutations in 14-3-3epsilon that affect signaling through Raf, impair the interaction of 14-3-3epsilon with PAR-1 (Benton, 2002).

Although many of the activities of PAR-1 kinases may be mediated by inducing 14-3-3 binding, this is probably not the only mechanism by which they act. Drosophila PAR-1 has recently been proposed to have a third function in the germline, in which it phosphorylates, and so stabilizes, OSK protein at the posterior pole of the oocyte to ensure its levels are high enough to specify the germ cells. Unlike PAR-1, 14-3-3 is not detectably enriched at the posterior, suggesting that this function of the kinase might operate via a 14-3-3-independent mechanism. C. elegans PAR-1 may have a similar function in germline specification, through the regulation of P-granule stability, which does not require PAR-5 (Benton, 2002).

A major question is the nature of the target(s) of PAR-1/14-3-3 that mediate their effects on cell polarity. These are unlikely to be any of the known PAR-1 substrates, such as Dishevelled or Tau, since these are not required for axis formation in Drosophila (R. B. and D. S. J., unpublished data reported in Benton, 2002), but the results lead to the clear prediction that they will bind to 14-3-3 in a PAR-1-dependent manner (Benton, 2002).

Interaction of Akt-phosphorylated Ataxin-1 with 14-3-3 mediates neurodegeneration in a Drosophila model of spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCA1) is one of several neurological disorders caused by a CAG repeat expansion. In SCA1, this expansion produces an abnormally long polyglutamine tract in the protein ataxin-1. Mutant polyglutamine proteins accumulate in neurons, inducing neurodegeneration, but an understanding of the mechanism underlying this accumulation has been unclear. The 14-3-3 protein, a multifunctional regulatory molecule, mediates the neurotoxicity of ataxin-1 by binding to and stabilizing ataxin-1, thereby slowing ataxin-1's normal degradation. The association of ataxin-1 with 14-3-3 is regulated by Akt phosphorylation, and in a Drosophila model of SCA1, both 14-3-3 and Akt modulate neurodegeneration. The finding that phosphatidylinositol 3-kinase/Akt signaling and 14-3-3 cooperate to modulate the neurotoxicity of ataxin-1 provides insight into SCA1 pathogenesis and identifies potential targets for therapeutic intervention (Chen, 2003).

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG repeat that produces an abnormally long polyglutamine tract in the ataxin-1 protein. At least eight other inherited neurodegenerative diseases, including Huntington's disease, are caused by a similar pathogenic mechanism. In each case, the length of the CAG repeat tract correlates with disease severity: more repeats produce more severe symptoms with an earlier age of onset. The expanded polyglutamine tract appears to confer a toxic gain-of-function that intensifies with longer repeats (Chen, 2003 and references therein).

Another feature common to the polyglutamine diseases studied so far (as well as several other neurodegenerative disorders) is aberrant protein deposition: mutant polyglutamine proteins have a strong tendency to accumulate and eventually form aggregates in neurons. It has been proposed that the polyglutamine expansion alters the protein's conformation in such a way as to make the protein recalcitrant to proteasomal degradation. In the case of ataxin-1, even the unexpanded protein can produce pathology if expressed at sufficiently high levels, which suggests that wild-type ataxin-1 might have more than one stable conformation, and that one or more of these alternate conformations is toxic if it becomes abundant. Support for this idea has come from the study of alpha-synuclein, whose accumulation causes Parkinson's Disease (PD). Although rare cases of familial PD are caused by point mutations in alpha-synuclein, most PD is associated with abnormal accumulation of wild-type alpha-synuclein. These observations raise several important questions: what factors contribute to the altered protein conformation? How exactly do misfolded proteins induce neuronal dysfunction and degeneration? And what factors modulate their toxicity (Chen, 2003 and references therein)?

The subcellular localization of the polyglutamine protein, the ratio of the polyglutamine tract to the host protein, and native protein sequences flanking the CAG repeat all affect the toxicity of polyglutamine proteins. Protein modifications such as phosphorylation may also have an effect: in Alzheimer's disease (AD): for example, brain dysfunction and degeneration are linked to the accumulation of the neurofibrillary tangles that are highly enriched in the hyperphosphorylated forms of the microtubule-associated protein tau. Enhanced phosphorylation of tau by glycogen synthase kinase 3beta (GSK3beta) induces filamentous tau inclusions and accelerates tau-induced neurodegeneration in transgenic flies and mice. Given these findings, it was asked whether protein phosphorylation might play a role in SCA1 pathogenesis as well (Chen, 2003 and references therein).

Ataxin-1 is phosphorylated at serine 776 (S776) and substitution of this S776 residue with alanine (A776) greatly diminishes the ability of mutant ataxin-1 to aggregate. These results suggest that a serine at position 776 of ataxin-1 plays a role in SCA1 pathogenesis. Because this serine is normally phosphorylated, it was speculated that S776 phosphorylation might modify ataxin-1 neurotoxicity by regulating its protein-protein interactions. To test this hypothesis, attempts were made to identify proteins that interact with ataxin-1-S776 but not ataxin-1-A776, to identify the kinase that phosphorylates S776 in ataxin-1, and to examine the effects of these factors on SCA1 pathogenesis (Chen, 2003).

14-3-3 proteins bind to phosphopeptide motifs in a variety of cellular proteins to regulate diverse biological processes such as signal transduction, cell cycle control, and apoptosis. The function of 14-3-3 binding to ataxin-1 remains unclear, since the cellular function of ataxin-1 is not well understood. The present study does, however, shed light on the mechanism by which 14-3-3 renders ataxin-1 more toxic to neurons (Chen, 2003).

14-3-3 can protect its target protein from proteolysis and dephosphorylation. For example, 14-3-3 stabilizes the nicotinic receptor alpha4 subunit, elevating its steady-state protein levels. In this study, 14-3-3 was found to bind and stabilize ataxin-1 and promote its accumulation in both transfected cells and transgenic flies. The ataxin-1/14-3-3 interaction might directly stabilize a conformation of ataxin-1 that resists degradation or it might impede access to other ataxin-1-interacting proteins that would facilitate protein clearance. Note that 14-3-3 interacts not only with the expanded mutant ataxin-1 but also the unexpanded wild-type protein. It is therefore possible that 14-3-3 regulates ataxin-1's clearance under physiological conditions. This regulation becomes problematic upon CAG repeat expansion, since longer polyglutamine tracts enhance ataxin-1's interaction with 14-3-3, further stabilizing the mutant protein (Chen, 2003).

14-3-3 promotes the accumulation of ataxin-1 and also enhances aggregate formation. The finding that 14-3-3 aggravates SCA1 pathogenesis together with data showing the absence of nuclear inclusions and neuronal dysfuction in mice overexpressing ataxin-1[82Q]-A776 might resurrect the old question of whether nuclear inclusions cause SCA1 pathogenesis, but when ataxin-1 is expressed at physiologic levels, under control of endogenous promoter, neuronal dysfunction occurs in the absence of visible nuclear inclusions. The absence of nuclear inclusions in ataxin-1[82Q]-A776 mice most likely results from efficient clearance of the mutant protein due to its lack of interaction with 14-3-3 (Chen, 2003 and references therein).

To investigate the possibility that sequestration of 14-3-3 with mutant ataxin-1 interferes with the cellular functions of 14-3-3, the effects of 14-3-3 overexpression on the SCA1 phenotype were evaluated in vivo and no evidence was found that loss of 14-3-3 cellular functions plays a major role in SCA1 pathogenesis. If SCA1 pathology is caused simply by sequestration of 14-3-3 by ataxin-1, one would expect exogenous 14-3-3 to suppress the phenotype -- yet overexpression of Drosophila 14-3-3epsilon in SCA1 flies aggravates degeneration. In fact, immunolabeling of cerebellar sections from transgenic mice overexpressing ataxin-1[82Q]-S776 reveals that the distribution of 14-3-3 remains grossly unchanged without sequestration into nuclear inclusions; the colocalization of the two proteins to inclusions in cell cultures could be modulated by differences in other cellular proteins or the nature of inclusions (formed over hours in cells versus days and weeks in mice). It is likely that 14-3-3 and ataxin-1 preferentially form soluble protein complexes in vivo, whereby only a minor fraction of 14-3-3 is present in nuclear aggregates (Chen, 2003).

Consistent with the notion that polyglutamine expansion confers some toxic gain-of-function onto the host protein, larger polyglutamine expansions in ataxin-1 were found to have a higher affinity for 14-3-3. 14-3-3 is able to stabilize wild-type ataxin-1, however, and overexpression of 14-3-3 in SCA130Q flies enhances the neurotoxicity of ataxin-1[30Q]. These observations are consistent with the proposed role for 14-3-3 in stabilizing ataxin-1. The neurotoxic effects of mutant ataxin-1 are likely to be more pronounced in cells expressing high levels of 14-3-3. Many of the 14-3-3 isoforms are abundantly expressed in brain tissue, with different expression patterns for each cell-type; isoforms beta, gamma, and nu are particularly abundant in Purkinje cells, which suffer the most severe degeneration. High expression levels of certain 14-3-3 isoforms could contribute to the selective neuronal vulnerability characteristic of SCA1 (Chen, 2003).

Previous studies have found links between 14-3-3 and other human neurodegenerative disorders. The neurofibrillary tangles in AD are composed primarily of hyperphosphorylated tau proteins and contain 14-3-3, which modulates tau phosphorylation. Whether this interaction stabilizes tau remains to be determined. In PD, 14-3-3 is detectable in Lewy bodies, which accumulate alpha-synuclein. Interestingly, alpha-synuclein shares sequence homology with 14-3-3 and binds both to 14-3-3 and to some 14-3-3 binding partners. This finding suggests a possible role for either 14-3-3 or 14-3-3 binding proteins in alpha-synuclein-induced pathology. Moreover, 14-3-3 was recently found to associate with alpha-synuclein in a soluble protein complex that mediates dopamine-dependent neurotoxicity. It would be interesting to determine whether 14-3-3 plays any role in stabilizing alpha-synuclein. When searching for consensus 14-3-3 binding motifs in other polyglutamine-containing proteins, the RXXSXP motif in ataxin-2, alpha1A subunit voltage-gated calcium channel, ataxin-7, and atrophin-1 was found. Further studies are necessary to determine if there is an interaction between these proteins and 14-3-3 and whether such interactions affect the pathogenesis of SCA2, SCA6, SCA7, and DRPLA, respectively (Chen, 2003 and references therein).

Akt phosphorylates ataxin-1 and promotes its binding to 14-3-3, which in turn leads to ataxin-1 accumulation and neurodegeneration. Loss of Drosophila Akt1 function suppresses ataxin-1-induced neurodegeneration in a dosage-dependent manner. Akt is activated when recruited to the plasma membrane and phosphorylated at T308 and S473 by PDK1 and a yet-to-be identified 'S473-kinase'. That Drosophila PI3K overexpression aggravates the SCA1 phenotype more than Drosophila Akt1 overexpression is consistent with the important role of Drosophila PI3K in fully activating the signaling cascade. Because Drosophila PDK1 overexpresssion is insufficient to promote ataxin-1-induced degeneration, it is proposed that the 'S473-kinase' plays a pivotal role in activating dAkt to modulate ataxin-1's toxicity (Chen, 2003).

PI3K/Akt signaling is a major pathway mediating survival signals in neuronal cells in response to factors such as insulin-like growth factor 1. Therefore, PI3K/Akt signaling is generally considered neuroprotective, acting against stress conditions that occur during neurodegeneration. IGF-1 is known to activate PI3K/Akt signaling and to protect against neuronal death induced by amyloid-beta peptide, a toxic agent in AD. Likewise, Akt activation triggered by IGF-1 inhibits neuronal death induced by mutant huntingtin (Chen, 2003 and references therein).

It is therefore surprising to find that in SCA1 flies, PI3K/Akt promotes ataxin-1-induced neurodegeneration. It is possible that PI3K and Akt not only trigger survival signaling, as they do under other conditions, but also induce ataxin-1 phosphorylation and thus its interaction with 14-3-3. Whatever survival-promoting effect they exert may be counteracted by the greater neurotoxicity of mutant ataxin-1 accumulation in the cells. It is unlikely that Akt phosphorylation of ataxin-1 was programmed solely as a self-destruction pathway to antagonize cell survival signaling; it is more likely that the physiological activity of ataxin-1 is regulated in accordance with cell survival signaling. The differential effects of PI3K/Akt signaling upon each pathogenic protein exemplify the diversity of cellular responses in different human neurodegenerative diseases. Activation of PI3K/Akt might have beneficial effects for some neurodegenerative diseases but be deleterious for others. The availability of fly and mouse models for various neurodegenerative disorders will allow in vivo analysis of PI3K/Akt signaling as well as 14-3-3 interactions in various neurodegenerative disorders. Because 14-3-3 proteins are functionally interchangeable in different species, data obtained in model organisms are likely to prove clinically relevant (Chen, 2003).

In sum, a mechanism has been found by which PI3K/Akt signaling and 14-3-3 modulate ataxin-1 neurotoxicity. The identification of factors modulating SCA1 pathology may lead to therapeutic interventions such as interfering with ataxin-1/14-3-3 interaction using small peptides or reducing PI3K/Akt signaling by specific kinase inhibitors (Chen, 2003).

Quantitative proteomic analysis of protein complexes: concurrent identification of interactors and their state of phosphorylation

Protein complexes have largely been studied by immunoaffinity purification and (mass spectrometric) analysis. Although this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components, identifying post-translational modifications, and detecting quantitative changes in complex composition or state of modification of complex components. A protocol has been developed that enables determination, in a single LC-MALDI-TOF/TOF analysis, the true protein constituents of a complex, to detect changes in the complex composition, and to localize phosphorylation sites and estimate their respective stoichiometry. The method is based on the combination of fourplex iTRAQ (isobaric tags for relative and absolute quantification) isobaric labeling and protein phosphatase treatment of substrates. It was evaluated on model peptides and proteins and on the complex Ccl1-Kin28-Tfb3 isolated by tandem affinity purification from yeast cells. The two known phosphosites in Kin28 and Tfb3 could be reproducibly shown to be fully modified. The protocol was then applied to the analysis of samples immunopurified from Drosophila melanogaster cells expressing an epitope-tagged form of the insulin receptor substrate homologue Chico. These experiments allowed identification 14-3-3ε;, 14-3-3zeta, and the insulin receptor as specific Chico interactors. In a further experiment, the immunopurified materials obtained from tagged Chico-expressing cells that were either treated with insulin or left unstimulated were cmpared. This analysis showed that hormone stimulation increases the association of 14-3-3 proteins with Chico and modulates several phosphorylation sites of the bait, some of which are located within predicted recognition motives of 14-3-3 proteins (Pflieger, 2008: Full text of article).

The two 14-3-3 proteins ε and zeta were identified as interactors of Chico, and their association appeared to increase upon insulin stimulation of cells. The mammalian homologues of Chico, IRS-1 as well as IRS-2 and IRS-4, were also shown to bind to 14-3-3 proteins. IRS-1 was proven to interact with 14-3-3β in 3T3L1 adipocytes, and this binding was shown to increase with insulin treatment. In contrast, another study did not observe a significant change of interaction between 14-3-3ε and IRS-1 upon hormonal stimulation in HepG2 cells; nevertheless this observation relied on Western blotting, which provides less accurate quantitative data than MS-based approaches and may not have been able to detect changes at or below 2-fold, such as those observed here using mass spectrometry techniques. In NIH-3T3 cells, 14-3-3ε was shown to interact with IRS-1 and protein kinase C-α, thus modulating insulin signaling and degradation. This study also observed an increased association of Chico and IR after a 7-min insulin treatment, which reflects activation of the insulin pathway involving tyrosine phosphorylation of Chico by IR (Pflieger, 2008).

Kc cells were stimulated with an insulin concentration and within a time window previously established to give a robust induction of the whole pathway. As a result, several insulin-dependent phosphosites, mainly phosphoserines, were identified, in Chico. The roles of phosphoserines/phosphothreonines in the mammalian homologue IRS-1 have been studied with regard to the regulation of the insulin pathway. Some serine residues, when phosphorylated, participate in the negative control of insulin signaling, whereas others appear to have a positive regulatory function. The homology of the Chico sequence to the mammalian IRS homologues is too weak to allow precise comparison of phosphosites. Nonetheless it is worth mentioning that some serine residues were shown previously to become partially or fully phosphorylated in rat and mouse IRS-1 after 5-min stimulation with 80-100 nM insulin, which is in agreement with the current observations. Among the phosphorylated residues identified in Chico, several appear to correlate with insulin stimulation either positively or negatively. Most interestingly, five sequences overlap with predicted recognition motives of 14-3-3 proteins. All but one of them were shown to become more highly phosphorylated upon stimulation, which correlates well with an enhanced association of the two 14-3-3 proteins with Chico. The differences of phosphorylation levels measured in samples Chico3 and Chico4 may be, at least in part, due to the different cell densities reached before induction. Despite differences in absolute phosphorylation levels, similar variations of the phosphorylation states (increase or decrease) were observed in the two samples upon insulin stimulation (Pflieger, 2008).

Phosphorylations on tyrosine residues were also expected at least upon insulin treatment. The presence of phosphotyrosine-containing peptides could not be conclusively established by the MS data. Nonetheless the intact protein Chico could be shown to contain phosphorylated tyrosines: a fraction of the samples Chico3 and Chico4 was analyzed by Western blot using an anti-phosphotyrosine antibody, and signal was detected in both insulin conditions with increased signal in the +INS case as expected (Pflieger, 2008).

Genome-wide phosphoacetylation of histone H3 at Drosophila enhancers and promoters

Transcription regulation is mediated by enhancers that bind sequence-specific transcription factors, which in turn interact with the promoters of the genes they control. This study shows that the JIL-1 kinase is present at both enhancers and promoters of ecdysone-induced Drosophila genes, where it phosphorylates the Ser10 and Ser28 residues of histone H3. JIL-1 is also required for CREB binding protein (CBP)-mediated acetylation of Lys27, a well-characterized mark of active enhancers. The presence of these proteins at enhancers and promoters of ecdysone-induced genes results in the establishment of the H3K9acS10ph and H3K27acS28ph marks at both regulatory sequences. These modifications are necessary for the recruitment of 14-3-3, a scaffolding protein capable of facilitating interactions between two simultaneously bound proteins. Chromatin conformation capture assays indicate that interaction between the enhancer and the promoter is dependent on the presence of JIL-1, 14-3-3, and CBP. Genome-wide analyses extend these conclusions to most Drosophila genes, showing that the presence of JIL-1, H3K9acS10ph, and H3K27acS28ph is a general feature of enhancers and promoters in this organism (Kellner, 2012).

Activation of transcription in higher eukaryotes requires the interaction between transcription factors bound to distal enhancers and proteins present at the promoter. Recent findings indicate that enhancers contain a variety of histone modifications that change during the establishment of specific cell lineages suggesting that these sequences may play a more complex role in transcription than previously thought. Given the presence of common as well as specific histone marks at enhancers and promoters, it is tempting to speculate that epigenetic modifications at these sequences serve to integrate various cellular signals required to converge in order to activate gene expression. Results described in this study support this hypothesis, demonstrating that the proteins that carry out these histone modifications are necessary to establish enhancer-promoter contacts and activate transcription of ecdysone-inducible genes (Kellner, 2012).

The execution of this process in Drosophila requires the recruitment of JIL-1 by mechanisms that are not well understood. Although the direct involvement of JIL-1 in the transcription process has been brought into question due to the failure to observe recruitment of JIL-1 to heat shock genes in polytene chromosomes, results presented in this study clearly indicate that JIL-1 affects transcription at different steps in the transcription cycle. At the promoter region, phosphorylation of H3S10 by JIL-1 results in the recruitment of 14-3-3 and, subsequently, histone acetyltransferases Elp3 and MOF (Karam, 2010). This study found that JIL-1 is also able to phosphorylate H3S28 at both promoters the enhancers. The establishment of the H3K9acS10ph and H3K27acS28ph modifications correlates with the recruitment of 14-3-3 to enhancers and promoters of ecdysone-induced genes. 14-3-3 has been implicated in numerous cellular processes, where it functions as a scaffold protein). 14-3-3 is found as dimers and multimers; each monomer is capable of binding two targets and can mediate and stabilize interactions between two phosphoproteins. Additionally, acetylation facilitates the dimerization of 14-3-3 molecules and their ability to bind certain substrates. Binding assays have demonstrated that 14-3-3 interacts weakly with H3 tail peptides phosphorylated at S10 and S28, but strong binding is detected if the peptide is both phosphorylated and acetylated on the neighboring lysine residues. Given the ability of 14-3-3 to serve as a scaffold for large protein complexes, its demonstrated interactions with H3K9acS10ph and H3K27acS28ph and the presence of these two modifications at enhancers and promoters, it is possible that contacts between these two sequences are stabilized by 14-3-3. This hypothesis is supported by 3C experiments indicating that induction of transcription of the Eip75B gene is accompanied by strong enhancer-promoter interactions. These interactions are lost in JIL-1, CBP, and 14-3-3 knockdown cells. Since these proteins act several steps downstream from transcription factor binding in the pathway leading to enhancer-promoter contacts, and loss of these proteins results in the abolishment of these contacts, it appears that these proteins, rather than specific transcription factors, may be responsible for enhancer promoter interactions at the ecdysone-inducible genes (Kellner, 2012).

Genome-wide studies using ChIP-seq clearly show the presence of JIL-1, H3K9acS10ph, and H3K27acS28ph at enhancers and promoters of most Drosophila genes. There is a clear correlation between the amount of JIL-1, H3K9acS10ph, and H3K27acS28ph at promoters and the level of transcripts associated with the gene. These three marks are also present at enhancers defined by the occurrence of H3K4me1 and H3K27ac, suggesting that the JIL-1 kinase is a regulator of histone dynamics at enhancers and promoters genome-wide. JIL-1, H3K9acS10ph, and H3K27acS28ph are found at low levels at enhancers before activation, which then increase in intensity and drop in baseline when found in combination with H3K27ac, a mark of active enhancers. These conclusions are different from those previously published examining the role of JIL-1 in transcription and dosage compensation (Regnard, 2011). This study concluded that JIL-1 binds active genes along their entire length and that the levels of JIL-1 are not associated with levels of transcription. The differences in the conclusions may be due to the different cell lines used -- male S2 cells versus female Kc cells -- and the emphasis of the analysis by Regnard on the expression of dosage-compensated genes in the male X-chromosome, which may contain JIL-1 throughout the genes as a consequence of their regulation at the elongation step. In addition, the study by Regnard used ChIP-chip on custom tiling arrays of the X chromosome plus cDNA arrays containing the whole genome. This strategy may bias the conclusions and suggest the presence of JIL-1 in the coding region of genes rather than at enhancers and promoters (Kellner, 2012).

Results presented in this study extend the previous list of histone modifications characteristic of active enhancers to include H3K9acS10ph and H3K27acS28ph. Enhancers tend to be cell type-specific and are determined during differentiation with the characteristic H3K4me1 modification. It is unclear how these regions are designated before activation and what keeps them in a poised state ready for activation upon receiving the proper signal from the cell. It is tempting to speculate that the presence of JIL-1 at enhancers prior to activation might play a role in maintaining the enhancer in this poised state. An important question for future studies is the mechanistic significance of the looping between enhancers and promoters in order to achieve transcription activation. One interesting possibility is that various signaling pathways in the cell contribute to building epigenetic signatures at enhancers and promoters in the form of histone acetylation and/or phosphorylation of various Lys/Ser/Thr residues. Acetylation marks at enhancers and promoters may then cooperate to recruit BRD4 (FS(1)H in Drosophila), which contains two bromodomains each able to recognize two different acetylated Lys residues. The requirement for acetylation of histones at enhancers and promoters in order to recruit Brd4 would ensure that several different signaling events have taken place before recruitment of P-TEFb by BRD4 can release RNAPII into productive elongation (Kellner, 2012).

A NudE/14-3-3 pathway coordinates dynein and the kinesin Khc73 to position the mitotic spindle

Mitotic spindle position is controlled by interactions of cortical molecular motors with astral microtubules. In animal cells, Partner of Inscuteable (Pins) acts at the cortex to coordinate the activity of Dynein and Kinesin-73 (Khc73; KIF13B in mammals) to orient the spindle. Though the two motors move in opposite directions, their synergistic activity is required for robust Pins-mediated spindle orientation. This study identified a physical connection between Dynein and Khc73 that mediates cooperative spindle positioning. Khc73's motor and MBS domains link Pins to microtubule plus ends, while its stalk domain is necessary for Dynein activation and precise positioning of the spindle. A motif in the stalk domain binds, in a phospho-dependent manner, 14-3-3ζ, which dimerizes with 14-3-3ε. The 14-3-3ζ/ε heterodimer binds the Dynein adaptor NudE to complete the Dynein connection. The Khc73 stalk/14-3-3/NudE pathway defines a physical connection that coordinates the activities of multiple motor proteins to precisely position the spindle (Lu, 2013).

Mitotic spindle orientation requires the coordination of several pathways that act on astral microtubules. These pathways may establish cortical-microtubule connections and generate the forces necessary for movement of this large cellular structure with metaphase spindle lengths varying from 2 mm in yeast to 60 mm of a Xenopus single-cell stage. The spindle-orientation protein Pins has a domain that has been thought to capture microtubules (Pins^linker), and another that generates force (Pins^TPR). This study attempted to understand how these two pathways function together by taking advantage of an induced polarity system in cultured S2 cells in which the two pathways can be selectively activated. This system allowed for the identification of the Khc73 stalk domain as a critical element that links Pins^TPR and Pins^linker pathways. This observation was used as a platform for establishing a complete physical connection between the two pathways. This study has also clarified the role of 14-3-3 proteins in spindle orientation, establishing that their interaction with Pins is likely to be indirect (through Dlg and Khc73) (Lu, 2013).

Khc73 performs two functions in Pins-mediated spindle positioning. First, it functions in the Pins^linker pathway to mediate cortical microtubule capture through its MBS and motor domains, respectively. The N-terminal portion of Khc73 is sufficient for linker activity, which is likely occurring through a Dlg^GK/Khc73^MBS interaction at the cortex and a microtubule/ Khc73^motor interaction at the spindle. This suggests that Khc73's motor domain could function at the cortex by itself, however, Ed:Khc73^motor did not have spindle positioning activity, indicating that other factors could be required or the motor domain is not functional in this context (e.g., as a monomer with the coiled-coil stalk). Khc73 must therefore rely on Dlg as an adaptor to target it to the cortex, which is where it can potentially function to facilitate the initial contact of astral microtubules (Lu, 2013).

Although Khc73's MBS domain directly interacts with Dlg, Khc73 is not seen to colocalize with cortical Pins, even though Dlg robustly localizes to Pins crescents. Instead, the motor protein is seen distinctly at the ends of microtubule, suggesting that Khc73 moves to the plus ends where it may be poised for capture by the cortical Pins^linker/Dlg complex. Thus, Khc73's N-terminal domains are likely to facilitate cortical microtubule capture by linking microtubule plus ends to cortical Dlg (Lu, 2013).

In addition to facilitating cortical microtubule capture, this study found that Khc73 also forms a physical connection to the Pins^TPR/Mud/Dynein pathway with its stalk region, which is essential for the synergistic function of the two pathways. Khc73 may activate Dynein by delivering NudE to the cortex, where Dynein is presumably localized by Pins^TPR/Mud. Although it is not possible to observe the localization of Dynein in S2 cells for technical reasons, there is good evidence that it is cortically localized by way of Pins^TPR/Mud. In HeLa cells, Dynein localizes to the cortex with the mammalian homolog of Mud, NuMA, along with mPins, during mitosis (Lu, 2013).

It is proposed that a 14-3-3 motif in Khc73's stalk region activates an 'idling' cortically localized Dynein by cargoing NudE. Interestingly, although the Khc73 14-3-3 motif mutant Khc73^S1374A has a distribution of spindle-orientation angles that isn't random, the distribution is bimodal such that the spindle angles are either fully aligned or orthogonal to the polarity axis. The bimodal phenotype is distinct from the Khc73^motor+MBS fragment, which has a canonical intermediate distribution of spindle angles, suggesting that there may be additional regions or domains in the stalk that are contributing to the bimodal phenotype. It is hypothesized that an element within Khc73's stalk region is required for the proper application of the forces generated from by two motor proteins to properly orient the mitotic spindle. Nevertheless, biochemical and genetic studies demonstrate that the 14-3-3 binding motif is, at the very least, required for proper Pins-mediated spindle positioning and required for Khc73's interaction with the 14-3-3 proteins and NudE (Lu, 2013).

Pins mediates spindle positioning by coordinating two motor proteins that, as a pair, facilitate the cortical capture of microtubules and also provide pulling forces to robustly orient the mitotic spindle. A model is proposed in which orientation occurs through an ordered series of events, beginning with the initial polarization of Pins, followed by recruitment of Mud through its Pins^TPR domain and Dlg through Pins^linker region. Cortical Mud then recruits cytoplasmic Dynein, which is not yet active and will remain inert, but poised at the cortex. Khc73 localizes to the plus ends of microtubules, where it establishes cortical-microtubule contacts through direct binding to Dlg and also delivers NudE to cortical Dynein, thereby activating it. As astral microtubules enter the proximity of the Dynein complex, Dynein can generate specifically timed cortical pulling forces necessary for robust spindle positioning. Future work will be directed at dissecting the precise timing of these synergistic events that underlie differentiation and tissue architecture (Lu, 2013).

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