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EVOLUTIONARY HOMOLOGS (part 2/2)

Profilin response to Rho family members and interaction with Diaphanous homologs

Rho small GTPase regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton. A protein, p140mDia, has been identified as a downstream effector of Rho. It is a mammalian homolog of Drosophila Diaphanous, a protein required for cytokinesis, and belongs to a family of formin-related proteins containing repetitive polyproline stretches. p140mDia binds selectively to the GTP-bound form of Rho and also binds to profilin. p140mDia, profilin and RhoA are co-localized in the spreading lamellae of cultured fibroblasts. They are also co-localized in membrane ruffles of phorbol ester-stimulated sMDCK2 cells that extend these structures in a Rho-dependent manner. The three proteins are recruited around phagocytic cups induced by fibronectin-coated beads. Their recruitment is not induced after Rho is inactivated by microinjection of botulinum C3 exoenzyme. Overexpression of p140mDia in COS-7 cells induces homogeneous actin filament formation. These results suggest that Rho regulates actin polymerization by targeting profilin via p140mDia beneath the specific plasma membranes (Watanabe, 1997).

The RHO1 gene encodes a homolog of mammalian RhoA small G-protein in the yeast S. cerevisiae. Rho1p is required for bud formation and is localized at a bud tip or a cytokinesis site. Bni1p is a potential target of Rho1p. Bni1p shares the FH1 and FH2 domains with proteins involved in cytokinesis or establishment of cell polarity. In S. cerevisiae, there is an open reading frame (YIL159W) that encodes another protein, BNR1 (BNI1 Related) having the FH1 and FH2 domains. Bnr1p interacts with another Rho family member, Rho4p, but not with Rho1p. Disruption of BNI1 or BNR1 does not show any deleterious effect on cell growth, but the bni1 bnr1 mutant shows a severe temperature-sensitive growth phenotype. Cells of the bni1 bnr1 mutant arrested at the restrictive temperature are deficient in bud emergence, exhibit a random distribution of cortical actin patches and often become multinucleate. These phenotypes are similar to those of the mutant of PFY1, which encodes profilin, an actin-binding protein. Yeast two-hybrid and biochemical studies demonstrate that Bni1p and Bnr1p interact directly with profilin at the FH1 domains. These results indicate that Bni1p and Bnr1p are potential targets of the Rho family members, interact with profilin and regulate the reorganization of actin cytoskeleton (Imamura, 1997).

As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Cdc12p is a member of a family of proteins including Drosophila Diaphanous, S. cerevisiae BNI1, and S. pombe fus1, all involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence cdc12p is shown to be located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homolog), cdc3 (profilin homolog), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. Based on these patterns, a model is presented in which ring assembly originates from a single point on the cortex; a molecular pathway for the functions of cytokinesis proteins is suggested. cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin (Chang, 1997).

Non-canonical Wnt signaling plays important roles during vertebrate embryogenesis and is required for cell motility during gastrulation. However, the molecular mechanisms of how Wnt signaling regulates modification of the actin cytoskeleton remain incompletely understood. The Formin homology protein Daam1 (see Drosophila DAAM) is important link between Dishevelled and the Rho GTPase for cytoskeletal modulation. Profilin1 is an effector downstream of Daam1 required for cytoskeletal changes. Profilin1 interacts with the FH1 domain of Daam1 and is localized with Daam1 to actin stress fibers in response to Wnt signaling in mammalian cells. In addition, depletion of Profilin1 inhibits stress fiber formation induced by non-canonical Wnt signaling. Inhibition or depletion of Profilin1 in vivo specifically inhibits blastopore closure in Xenopus but does not affect convergent extension movements, tissue separation or neural fold closure. These studies define a molecular pathway downstream of Daam1 that controls Wnt-mediated cytoskeletal reorganization for a specific morphogenetic process during vertebrate gastrulation (Sato, 2006).

Other proteins that interact with profilin

The binding to poly(L-proline) is used for the affinity purification of profilins, but little is known about the structural and thermodynamic aspects of the interaction. Although a 6 residue type II poly(L-proline) helix can span the binding site, highest affinity binding is achieved by proline oligomers > or = 10 residues. Binding is stereospecific since (D-proline)11 does not bind. In 75 mM KCI the dissociation equilibrium constant for poly(L-proline) is about 10 microM proline decamer units for amoeba profilin and 20-30 microM for human profilin. Consistent with a significant hydrophobic component of the interaction, delta Cp is negative; higher salt concentrations enhance the affinity. No protons dissociate or bind during the interaction. Binding of poly(L-proline) is favored both entropically and enthalpically. Substitution of glycine in proline undecamers reduces affinity by about 1 kcal mol-1 for each substitution due to increased rotational freedom of the free peptides. Substitution of alanine has a similar effect. Disorder in the free peptides imparts an unfavorable entropic cost for immobilizing the substituted peptides on the binding site on profilin (Petrella, 1996).

Annexin I belongs to a family of calcium-dependent phospholipid-binding and membrane-binding proteins. Although many of the biochemical properties and the three-dimensional structure of this protein are known, its true physiological roles have yet to be thoroughly defined. Its putative functions include participation in the regulation of actin microfilament dynamics, proposed after the discovery of an interaction with actin. In accordance with this hypothesis, annexin I was also found to interact with profilin. The affinity of annexin I for profilin is between 10(7) M and 10(8) M. Calcium, a modulator of annexin I functions interfers only marginally with the association. Proteins or compounds known to interact with annexin I or profilin are found to inhibit the annexin-I--profilin interaction when added in the reaction medium. Recombinant profilin exhibits a slightly lower affinity than natural platelet protein when measured. Due to the submembrane localization of annexin I and the regulatory activity of profilin on the cytoskeleton, an interaction between annexin I and profilin may therefore be implicated in the regulation of some cellular functions, particularly those governing membrane-cytoskeleton dynamic organization (Alvarez-Martinez, 1996).

Profilins bind to monomeric actin and also interact with ligands such as phosphoinositide 4,5-bisphosphate, the proline-rich protein VASP and a complex of four to six polypeptides identified in Acanthamoeba that includes two actin-related proteins. A mutation in an essential gene from Schizosaccharomyces pombe, sop2+, rescues the temperature-sensitive lethality of a profilin mutation, cdc3-124. The sop2-1 mutant is defective for cell elongation and septation, suggesting that it is involved in multiple cortical actin-requiring processes. Consistent with a role in actin cytoskeletal function, negative interactions have been identified between sop2-1 and act1-48, a mutant allele of actin. Sop2p is a novel 377 amino acid polypeptide with similarity to proteins of the beta-transducin repeat family. Sop2p-related proteins have been identified by sequencing projects in diverse species; a human cDNA highly related to sop2+, SOP2 Hs, has been isolated. This protein functionally complements the sop2-1 mutation. Sop2p proteins from all species contain peptide sequences identical or highly similar to two peptide sequences from an Acanthamoeba beta-transducin repeat protein present in the profilin binding complex. Biochemical analyses demonstrate that Sop2p is present in a complex that also contains the actin-related protein, Arp3p. Sop2p is present in (1) punctate structures distributed throughout the cell, (2) cables that extend the length of the cell, and (3) a medial band in a small percentage of septating cells. Collectively these data demonstrate the interaction of Sop2p with Arp3p, profilin and actin (Balasubramanian, 1996).

Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice, profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching a number of ligands for profilin I and profilin II from mouse brain extracts were characterized, including dynamin I, clathrin, synapsin, Rho-associated coiled-coil kinase, the Rac-associated protein NAP1 and a member of the NSF/sec18 family. In vivo, profilins co-localize with dynamin I and synapsin in axonal and dendritic processes. These findings strongly suggest that in the brain, profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains (Witke, 1998).

N-WASP, which was first characterized as a protein that binds an adaptor protein Ash/Grb2 through its SH3 domain, has many proline-rich sequences, suggesting some interaction with profilin other than through Ash/Grb2. N-WASP functions as an effector of activated-Cdc42 in filopodium formation. Furthermore, N-WASP has Cdc42-dependent actin-depolymerizing activity in its cofilin homology domain that seems to be necessary for the creation of barbed ends. However, the role of N-WASP in microspike extension including actin polymerization, has been unclear. A profilin mutant (H119E) has been prepared that is defective in actin binding, but retains the ability to bind to other proteins. This mutant profilin I suppresses actin polymerization in microspike formation induced by N-WASP, the essential factor in microspike formation. Profilin associates both in vivo and in vitro with N-WASP at proline-rich sites different from those to which Ash/Grb2 binds. This association between profilin and N-WASP is required for N-WASP-induced efficient microspike elongation. Moreover, microspike formation in permeabilized cells can be reconstituted using profilin I combined with N-WASP and its regulator, Cdc42. These findings provide the first evidence that profilin is a key molecule linking a signaling network to rapid actin polymerization in microspike formation (Suetsugu, 1998).

Mammalian enabled (Mena) is a member of a protein family thought to link signal transduction pathways to localized remodeling of the actin cytoskeleton (See Drosophila Enabled). Mena binds directly to Profilin, an actin-binding protein that modulates actin polymerization. The distribution of Mena in wild-type adult organs was compared to that of EVL (Ena-VASP-like) and VASP. The 140 kDa form of Mena is detected only in the brain, while the 80 kDa form of Mena is expressed predominantly in brain, testis, ovaries, and fat. In contrast, EVL and VASP are most highly expressed in thymus and spleen, and the relative intensities of the phospho and dephospho forms vary from tissue to tissue, suggesting that EVL and VASP may be differentially regulated in the brain and organs. Because Mena is expressed at high levels in the brain, while both VASP and EVL are expressed at low levels, the distribution of Mena in adult and developing brain was characterized in greater detail. The 80 and 140 kDa forms of Mena are detected in all regions of the adult brain, with highest levels in the hippocampus, cortex, and midbrain, and lowest levels in the striatum and cerebellum. The 140 kDa form of Mena is expressed at relatively high levels in serum-free cortical cultures (which are enriched for neurons) and is not detected in glial cultures, suggesting the 140 kDa form is indeed neuron-specific and that it may be the predominant form of Mena in neurons. In embryonic brains, all three forms of Mena are detected at embryonic day 11 (E11), the earliest time point examined. Expression of the 88 kDa form decreases steadily and becomes almost undetectable by E16, while expression of the 140 kDa form begins to increase at E13 and peaks between E16 and E18. In contrast to Mena, EVL expression in the brain is first detected at E15. VASP expression appears to be fairly constant throughout development of the brain, but then decreases to relatively low levels in the adult brain (Lanier, 1999).

At E8.5, Mena is particularly enriched in the neuroepithelium, the forebrain, and the somites. Mena is highly expressed in the edges of the neural folds. By E10.5, Mena expression is detected in the brain, dorsal root ganglia (DRG), somites, and limb buds. In addition, Mena is highly expressed in the branchial and pharyngeal arches, neural crest-derived structures that give rise to portions of the face and neck. In primary neurons, Mena is concentrated at the tips of growth cone filopodia. High levels of Mena expression are detected in distinct bands of cells in the developing cortex at E16, a time when neurons are migrating from the ventricular zone to the cortical layers and axons are beginning to project across the corpus callosum. In the adult brain, Mena expression is detected in laminae 2/3 and 5 of the cortex and is particularly enriched in the hippocampus and the septum. In neurons, Mena is highly enriched in the lamellipodium and at the tips of the axonal growth cones. Similar Mena localization is seen in dendritic growth cones and at various stages of differentiation (Lanier, 1999)

Mena-deficient mice are viable; however, axons projecting from interhemispheric cortico-cortical neurons are misrouted in early neonates, and fail decussation of the corpus callosum. Fibers in the corpus callosum appear to reach a point just medial to the cingulum bundle as normal but then failed to project medially and cross the midline. Defects in the hippocampal commissure and the pontocerebellar pathway are evident in the adult. Mena-deficient mice that are heterozygous for a Profilin I deletion die in utero and display defects in neurulation, demonstrating an important functional role for Mena in regulation of the actin cytoskeleton. The neural tube closure defects seen in the Mena;profilin mutant animals reveal that Mena plays a critical role in neurulation in addition to its function in axon guidance. Mena function in neurulation involves Profilin and therefore might be linked to regulation of the actin cytoskeleton. Cephalic neural tube closure depends on actin-driven changes in the shape of cells within the dorsal-lateral hinge point of the neuroepithelium (Lanier, 1999).

Gephyrin is an essential component of the postsynaptic cortical protein network of inhibitory synapses. Gephyrin-based scaffolds participate in the assembly as well as the dynamics of receptor clusters by connecting the cytoplasmic domains of glycine and GABA(A) receptor polypeptides to two cytoskeletal systems: microtubules and microfilaments. Although there is evidence for a physical linkage between gephyrin and microtubules, the interaction between gephyrin and microfilaments is not well understood so far. Neuronal gephyrin has been shown to interact directly with key regulators of microfilament dynamics, profilin I and neuronal profilin IIa, and with microfilament adaptors of the mammalian enabled (Mena)/vasodilator stimulated phosphoprotein (VASP) family, including neuronal Mena. Profilin and Mena/VASP coprecipitate with gephyrin from tissue and cells, and complex formation requires the E-domain of gephyrin, not the proline-rich central domain. Consequently, gephyrin is not a ligand for the proline-binding motif of profilins, as suspected previously. Instead, it competes with G-actin and phospholipids for the same binding site on profilin. Gephyrin, profilin, and Mena/VASP colocalize at synapses of rat spinal cord and cultivated neurons and in gephyrin clusters expressed in transfected cells. Thus, Mena/VASP and profilin can contribute to the postulated linkage between receptors, gephyrin scaffolds, and the microfilament system and may regulate the microfilament-dependent receptor packing density and dynamics at inhibitory synapses (Giesemann, 2003).

The small GTPase Rap1 induces integrin-mediated adhesion and changes in the actin cytoskeleton. The mechanisms that mediate these effects of Rap1 are poorly understood. RIAM was identified as a Rap1-GTP-interacting adaptor molecule. RIAM defines a family of adaptor molecules that contain a RA-like (Ras association) domain, a PH (pleckstrin homology) domain, and various proline-rich motifs. The protein with highest homology to RIAM is Lamellipodin (Krause, 2004). Furthermore, RIAM is related to proteins CG11940 (AAF49029) in D. melanogaster and Mig-10 (P34400) in C. elegans. RIAM interacts with Profilin and Ena/VASP proteins, molecules that regulate actin dynamics. Overexpression of RIAM induces cell spreading and lamellipodia formation, changes that require actin polymerization. In contrast, RIAM knockdown cells have reduced content of polymerized actin. RIAM overexpression also induces integrin activation and cell adhesion. RIAM knockdown displaces Rap1-GTP from the plasma membrane and abrogates Rap1-induced adhesion. Thus, RIAM links Rap1 to integrin activation and plays a role in regulating actin dynamics (Lafuente, 2004).

Profilins, regulators of cytoplasmic actin dynamics, also bind to several nuclear proteins but the significance of these interactions is mostly unclear. A novel Myb-related transcription factor, p42(POP), is described as a new ligand for profilin and profilin is shown to regulate its activity. p42(POP) comprises a unique combination of domains and is widely expressed in mouse tissues. In contrast to many other Myb proteins, it contains only one functional tryptophan-cluster motif. This is followed by an acidic domain, a leucine zipper that mediates dimerization and functional nuclear import and export signals that can direct p42(POP) to either the nuclear or the cytoplasmic compartment. Binding to profilins is mediated by a proline-rich cluster. p42(POP)-profilin complexes can be precipitated from cell lysates. In transfected cells displaying p42(POP) in the nucleus, nuclear profilin is markedly increased. When p42(POP) is anchored at mitochondrial membranes, profilin is targeted to this location. Hence, in a cellular environment, p42(POP) and profilin are found in the same protein complex. In luciferase assays, p42(POP) acts as repressor and this activity is substantially reduced by profilins, indicating that profilin can regulate p42(POP) activity and is therefore involved in gene regulation (Lederer, 2005).

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