myoblast city
During programmed cell death, cell corpses are rapidly engulfed. This engulfment process involves the
recognition and subsequent phagocytosis of cell corpses by engulfing cells. How cell corpses are
engulfed is largely unknown. ced-5, a gene that is required for cell-corpse
engulfment in the nematode C. elegans, encodes a protein that is similar to the human
protein DOCK180 and the Drosophila protein Myoblast City (Mbc), both of which have
been implicated in the extension of cell surfaces. ced-5 mutants are defective not only in the
engulfment of cell corpses but also in the migrations of two specific gonadal cells, the distal tip cells.
The expression of human DOCK180 in C. elegans rescues the cell-migration defect of a ced-5 mutant. Evidence is presented that ced-5 functions in engulfing cells during the engulfment of cell corpses. It is
suggested that ced-5 acts in the extension of the surface of an engulfing cell around a dying cell during
programmed cell death. This new family of proteins that function in the extension of cell
surfaces have been named the CDM (for CED-5, DOCK180 and MBC) family (Wu, 1998).
The C. elegans genome contains three rac-like genes: ced-10, mig-2, and rac-2. ced-10, mig-2 and rac-2 act redundantly in axon pathfinding: inactivating one gene has little effect, but inactivating two or more genes perturbes both axon outgrowth and guidance. mig-2 and ced-10 also have redundant functions in some cell migrations. By contrast, ced-10 is uniquely required for cell-corpse phagocytosis, and mig-2 and rac-2 have only subtle roles in this process. Rac activators are also used differentially. The UNC-73 Trio Rac GTP exchange factor affects all Rac pathways in axon pathfinding and cell migration but does not affect cell-corpse phagocytosis. CED-5 DOCK180, which acts with CED-10 Rac in cell-corpse phagocytosis, acts with MIG-2 but not CED-10 in axon pathfinding. Thus, distinct regulatory proteins modulate Rac activation and function in different developmental processes (Lundquist, 2001).
The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. An
additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2, are described. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals (Gumienny, 2001).
CRK belongs to a family of adaptor proteins that consist mostly of SH2 and SH3 domains. Far
Western blotting with CRK SH3 has demonstrated that it binds to a number of proteins. A 135-145 kDa protein is C3G, a CRK SH3-binding guanine nucleotide exchange
protein. The molecular cloning of the 180 kDa protein, which is designated
DOCK180 (a 180 kDa protein downstream of CRK) is reported. The isolated cDNA contains a 5,598-bp open
reading frame encoding a 1,866-amino-acid protein. The deduced amino acid sequence does not reveal
any significant homology to known proteins, except that an SH3 domain is identified at its amino
terminus. To examine the function of DOCK180, a Ki-Ras farnesylation signal was fused to the
carboxyl terminus of DOCK180, a strategy that has been employed successfully for activation of
adaptor-binding proteins in vivo. Whereas wild-type DOCK180 accumulates diffusely in the cytoplasm
and does not have any effect on cell morphology, farnesylated DOCK180 localizes on the
cytoplasmic membrane and changes spindle 3T3 cells to flat, polygonal cells. These results suggest that
DOCK180 is a new effector molecule, which transduces signals from tyrosine kinases through the CRK adaptor protein (Hasegawa, 1996).
CRK is a human homolog of chichen v-Crk, which is an adaptor protein. The SH2 domain of CRK binds to several tyrosine-phosphorylated proteins, including the epidermal growth factor receptor, p130(Cas), Shc, and paxillin. The SH3 domain, in turn, binds to multiple cytosolic proteins. Expression libraries were screened by far western blotting, using CRK SH3 as a probe, and partial cDNA sequences of four distinct proteins were identified, including C3G, DOCK180, EPS15, and
clone ST12. The consensus sequence of the CRK SH3 binding sites, as deduced from their amino acid sequences, is Pro+3-Pro+2-X+1-Leu0-Pro-1-X-2-Lys-3. The interaction of the CRK SH3 domain with the DOCK180 peptide was examined with an optical biosensor, based on the principles of surface plasmon resonance. A low dissociation constant of the order of 10(-7) resulted from a high association rate constant and low dissociation rate. All CRK-binding proteins except clone ST12 also bind to another adaptor protein, Grb2. Mutational analysis reveals that glycine at position +1 of ST12 inhibits the binding to Grb2 while retaining the high affinity binding to CRK SH3. The result suggests that the amino acid at position +1 also contributes to the high affinity binding of the peptides to the SH3 domain of Grb2, but not to that of CRK (Matsuda, 1996).
The observation that DOCK180 associates equally well with activated or
dominant-negative forms of Rac suggests that, like the guanine
nucleotide exchange actors (GEFs), this interaction may be mediated by
the nucleotide-free form of the GTPase. To test this
possibility, the interaction of DOCK180 with bacterially
expressed GST-Rac was examined in the presence or absence of nucleotide. The
interaction of DOCK180 with Rac is blocked by the addition of either
GDP or GTP gammaS, suggesting that DOCK180 preferentially
forms a complex with the nucleotide-free form of Rac. Additionally, no
association between DOCK180 and nucleotide-free Rho is observed. The
interaction of the Rho/Rac effector PRK2 with Rac is
detected only in the presence of active, GTPgammaS-bound Rac, confirming that
nucleotide-dependent binding can be detected in this assay (Nolan, 1998 and references).
The observation that DOCK180 can be found in a complex with
nucleotide-free Rac strongly suggests that Mbc/DOCK180 is
functioning upstream of Rac. In cultured cells expressing activated
Rac, an increase in JNK activity can be readily detected. Therefore, to determine whether DOCK180 could contribute to Rac activation, JNK activity was examined in DOCK180-transfected mammalian cells. Cos cells were transfected with c-Jun together with RacV12, DOCK180, or DOCK180 and RacN17, and c-Jun phosphorylation was measured by immunoblotting with an anti-phospho-c-Jun antibody. Anisomycin strongly induces phosphorylation of c-Jun. DOCK180 stimulates JNK activity to an extent similar to that of stimulation by RacV12, and this stimulation of JNK activity is blocked by coexpression of RacN17. This result indicates that DOCK180 is likely to function upstream of Rac (Nolan, 1998).
Because Mbc/DOCK180/CED-5 proteins do not contain
the Dbl-homology domain found in all known GEFs for the Rho family of
GTPases, it is unlikely that this group of proteins functions
directly as a Rac activator. The Rac-DOCK180-binding results described above do not exclude the presence of additional components that bridge this
interaction. DOCK180 binds to the adaptor protein Crk (Hasegawa. 1996), which associates directly with the Rac GEFs, Sos and Vav, via its amino-terminal SH3
domain. Although the interaction of DOCK180 with Crk requires this same SH3 domain, implying that these are mutually exclusive complexes, it
was reported recently that p130 CAS (Crk-associated substrate), which
contains multiple SH2-dependent Crk-binding sites,
regulates cell migration in a Rac-dependent manner (Klemke,
1998). Possibly CAS serves as a scaffold for multiple Crk complexes,
some that include DOCK180 and others that include Rac GEFs, and this
complex facilitates the interaction of Rac with the Rac GEFs, thereby
leading to Rac activation. Interestingly, both Crk and CAS localize to
membrane ruffles in migratory cells (Klemke, 1998), raising the
possibility that a complex containing Crk and CAS as well as a Rac GEF
and DOCK180 leads to subcellularly localized Rac activation. Since
targeting of DOCK180 to the plasma membrane causes morphological
changes that resemble those seen in response to Rac activation
(Hasegawa, 1996), it is possible that the role of DOCK180 in such
a complex is to facilitate localization of these proteins to cell membranes (Nolan, 1998).
DOCK180 is one of the two principal proteins bound to the SH3 domain of the adaptor protein CrkII. The involvement of DOCK180 in integrin signaling has been studied. DOCK180 is neither phosphorylated nor bound to CrkII in quiescent NIH 3T3 cells and 3Y1 cells. DOCK180 is phosphorylated and bound to CrkII in NIH 3T3 cells stimulated with integrin and also in 3Y1 cells transformed by v-src or v-crk. The binding of DOCK180 to CrkII correlates with the binding of CrkII to p130(Cas), which is a major CrkII SH2 domain-binding protein at focal adhesions.
In a reconstitution experiment, expression of DOCK180 induces hyperphosphorylation of p130(Cas) and a concomitant increase in the amount of CrkII bound to p130(Cas). Similarly, binding of DOCK180 to CrkII is also enhanced by the coexpression of p130(Cas). Coexpression of p130(Cas) and CrkII with DOCK180 induces local membrane spreading and accumulation of DOCK180-CrkII-p130(Cas) complexes at focal adhesions. These findings suggest that DOCK180 positively regulates signaling from integrins to CrkII-p130(Cas) complexes at focal adhesions (Kiyokawa, 1998a).
DOCK180 is involved in integrin signaling through CrkII-p130Cas complexes (see CAS/CSE1 segregation protein). The involvement of DOCK180 in Rac1 signaling cascades has been studied. Both Rac1 and Cdc42Hs are known to activate JNK [Jun (amino) N-kinase]. DOCK180 activates JNK in a manner dependent on Rac1, Cdc42Hs, and SEK, and overexpression of DOCK180 increases the amount of GTP-bound Rac1 in 293T cells. GST-tagged JNK is expressed in 293T cells with or without DOCK180. The in vitro kinase activity of JNK has been examined by use of c-Jun as a substrate. JNK is activated by DOCK180 to a similar extent as the constitutively active Cdc42HsQL mutant. Another MAP kinase, ERK, is not activated by DOCK180. Coexpression of dominant-negative mutants of Rac1 (RacN17), Cdc42Hs (Cdc42N17), or SEK (SEKDN) inhibits DOCK180-dependent activation of JNK. Thus, DOCK180 appears to activate the JNK pathway in a manner dependent on Rac1, Cdc42Hs, and SEK. Furthermore, activation of JNK by DOCK180 is enhanced by the expression of CrkII, suggesting that signaling from tyrosine kinases may activate JNK through the CrkII-DOCK180 complex. The basal level of GTP-bound Rac1 is ~2.0% in 293T cells. Coexpression of DOCK180 increases the GTP-bound Rac1 to 6.2%. Direct binding of DOCK180 to Rac1 is observed, but not binding to RhoA or Cdc42Hs. Cultured cells were transfected with expression vectors, lysed, and GST-tagged proteins were collected on glutathione-Sepharose and examined for binding to DOCK180 by immunoblotting. DOCK180 coprecipitates with Rac1 only when cells are lysed in EDTA-containing buffer. Binding of Rac1 to DOCK180 in the presence of EDTA strongly suggests that DOCK180 binds to the nucleotide-free Rac1 protein. To avoid possible artifacts arising from the use of GST-tagged proteins and to understand the role of guanine nucleotides on Rac1/DOCK180 association, HA-tagged Rac1 proteins (RacN17 and RacV12) were used. Both RacN17 and RacV12 bind to DOCK180 in the presence of EDTA when coexpressed in cultured cells. However, only RacN17 binds to DOCK180 when Mg2+ is included in the lysis buffer. RacN17 binds to guanine nucleotides less efficiently than does the wild-type Rac1; therefore, this also implies that DOCK180 binds to guanine nucleotide-free Rac1. Only Rac1 binds to DOCK180; the nucleotide-free Rac1, but neither GTP-S- nor GDP-loaded Rac bind to DOCK180. Dominant-negative Rac1 suppresses DOCK180-induced membrane spreading. These results strongly suggest that DOCK180 is a novel activator of Rac1 and involved in integrin signaling. It has been shown that CrkII-p130Cas complexes regulate cell spreading after integrin stimulation and serve as a molecular switch for induction of cell migration. However, the downstream signaling molecules of CrkII have not been identified. There is substantial evidence that DOCK180 is the downstream effector of CrkII in integrin signaling (Kiyokawa, 1998): (1) DOCK180 binds to CrkII after integrin stimulation; (2) DOCK180 colocalizes with the CrkII-p130Cas complexes at focal adhesions and at the sites of membrane spreading, and (3) the expression of DOCK180 in 293T cells accelerates the formation of the CrkII-p130Cas complexes. Because Rac1 is also known to be involved in cell migration and spreading, it is likely that the DOCK180 recruited to the CrkII-p130Cas complexes on integrin stimulation transduces signals to Rac1 at focal adhesions, which eventually induces cell spreading (Kiyokawa, 1998b).
Engulfment of apoptotic cells in C. elegans is controlled by two partially redundant pathways. Mutations in genes in one of these pathways, defined by the genes ced-2, ced-5 and ced-10, result in defects both in the engulfment of dying cells and in the migrations of the two distal tip cells of the developing gonad. ced-2 and ced-10 encode proteins similar to the human adaptor protein CrkII and the human GTPase Rac, respectively. Together with the previous observation that ced-5 encodes a protein similar to human DOCK180, these findings define a signaling pathway that controls phagocytosis and cell migration. Evidence is provided that CED-2 and CED-10 function in engulfing rather than dying cells to control the phagocytosis of
cell corpses, that CED-2 and CED-5 physically interact, and that ced-10 probably functions downstream of ced-2 and ced-5. It is proposed that CED-2/CrkII and CED-5/DOCK180 function to activate CED-10/Rac in a GTPase signaling pathway that controls the polarized extension of cell surfaces (Reddien, 2000).
Integrin receptors are important for the phagocytosis of apoptotic cells.
However, little is known about their function in mediating internalization, since
previous studies have used blocking antibodies for the inhibition of binding. The alphavbeta5 receptor mediates both binding and internalization of
apoptotic cells. Internalization is dependent upon signaling through the beta5
cytoplasmic tail, and engagement of the alphavbeta5 heterodimer results in
recruitment of the p130cas-CrkII-Dock180 molecular complex, which in turn
triggers Rac1 activation and phagosome formation. In addition to defining
integrin-receptor signaling as critical for the internalization of apoptotic
material, these results also constitute the first evidence in human cells that the CED-2-CED-5-CED-10 complex defined in C. elegans is functionally
analagous to the CrkII-Dock180-Rac1 molecular complex in mammalian cells. By
linking the alphavbeta 5 receptor to this molecular switch, an
evolutionarily conserved signaling pathway has been revealed that is responsible for the recognition and internalization of apoptotic cells by both professional and non-professional phagocytes (Albert, 2000).
Nck-2 is a newly identified adapter protein comprising three N-terminal SH3
domains and one C-terminal SH2 domain. In a yeast two-hybrid
screen, DOCK180, a signaling protein implicated in the regulation of membrane
ruffling and migration, has been identified as a binding protein for Nck-2. Surface plasmon resonance analyses reveal that the second and the third SH3 domains interact with the C-terminal region of DOCK180. The interactions mediated by the individual SH3 domains, however, are much weaker than those of the full length Nck-2. Furthermore, a point mutation that inactivates the second or the third SH3 domain dramatically reduces the interaction of Nck-2 with DOCK180,
suggesting that both SH3 domains contribute to the DOCK180 binding. A major
Nck-2 binding site, which is recognized primarily by the third SH3 domain, has
been mapped to residues 1819-1836 of DOCK180. Two additional, albeit much
weaker, Nck-2 SH3 binding sites are located to DOCK180 residues 1793-1810 and
1835-1852, respectively. Consistent with the mutational studies, kinetic analyses
by surface plasmon resonance suggest that two binding events mediate
the binding of GST-Nck-2 to GST fusion protein containing the C-terminal region
of DOCK180. These studies identify a novel interaction between Nck-2 and DOCK180. Furthermore, they provide a detailed analysis of a protein complex formation mediated by multiple SH3 domains revealing that tandem SH3 domains significantly enhance the weak interactions mediated by each individual SH3 domain (Tu, 2001).
The alpha(5) chain-containing laminin isoforms, laminins-10 and -11
(laminin-10/11), are the major components of the basement membrane, having
potent cell-adhesive activity. The cell-adhesive and
integrin-mediated signaling activities of laminin-10/11 have been examined in comparison with fibronectin, the best characterized extracellular adhesive ligand. Laminin-10/11 are more active than fibronectin in promoting cell migration and
preferentially activate Rac, not Rho, via the p130(Cas)-CrkII-DOCK180 pathway.
Cells adhering to fibronectin develop stress fibers and focal contacts, whereas
cells adhering to laminin-10/11 do not, consistent with the high cell
migration-promoting activity of laminin-10/11. Pull-down assays of GTP-loaded
Rac and Rho demonstrate the preferential activation of Rac on laminin-10/11, in
contrast to the activation of Rho on fibronectin. Activation of Rac by
laminin-10/11 is associated with the phosphorylation of p130(Cas) and an
increased formation of a p130(Cas)-CrkII-DOCK180 complex. Cell migration on
laminin-10/11 is suppressed by the expression of either a dominant-negative Rac
or CrkII mutant defective in p130(Cas) or DOCK180 binding. This is the first
report demonstrating a distinct activation of Rho family GTPases resulting from
adhesion to different extracellular ligands (Gu, 2001).
DOCK180 was originally identified as one of two major proteins bound to the Crk oncogene product and became an archetype of the CDM family of proteins,
including Ced-5 of C. elegans and Mbc of Drosophila.
Further study has suggested that DOCK180 is involved in the activation of Rac by
the CrkII-p130(Cas) complex. With the use of deletion mutants of DOCK180, it has been found that the C-terminal region containing a cluster of basic amino acids is required for binding to and activation of Rac. This region shows high
amino-acid sequence similarity to the consensus sequence of the
phosphoinositide-binding site; this led to an examination of whether this basic region binds to phosphoinositides. For this purpose PtdIns(3,4,5)P(3)-APB
beads were used. By using various competitors, the specific binding of DOCK180 to PtdIns(3,4,5)P(3) has been demonstrated. The expression of active
phosphoinositide 3-kinase (PI-3K) does not enhance a DOCK180-induced increase in
GTP-Rac; however, the expression of PI-3K translocates DOCK180 to the plasma
membrane. Thus DOCK180 contains a phosphoinositide-binding domain, as do the
other guanine nucleotide exchange factors with a Dbl homology domain, and is
translocated to the plasma membrane on the activation of PI-3K (Kobayashi, 2001).
Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. A domain within Dock180 (denoted Docker) has been identified that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180 ELMO1 interaction is required. A trimeric ELMO1 Dock180 Rac1 complex and ELMO augments the interaction between Dock180 and Rac. It is proposed that the Dock180 ELMO complex functions as an unconventional two-part exchange factor for Rac (Brugnera, 2002).
Mammalian DOCK180 protein and its orthologues Myoblast City (MBC) and CED-5 in Drosophila and Caenorhabditis elegans, respectively, function as critical regulators of the small GTPase Rac during several fundamentally important biological processes, such as cell motility and phagocytosis. The mechanism by which DOCK180 and its orthologues regulate Rac has remained elusive. A domain within DOCK180 named DHR-2 (Dock Homology Region-2) has been identified that specifically binds to nucleotide-free Rac and activates Rac in vitro. These studies further demonstrate that the DHR-2 domain is both necessary and sufficient for DOCK180-mediated Rac activation in vivo. Importantly, several novel homologues of DOCK180 have been identified that possess this domain and many of them are found to directly bind to and exchange GDP for GTP both in vitro and in vivo on either Rac or another Rho-family member, Cdc42. These studies therefore identify a novel protein domain that interacts with and activates GTPases and suggest the presence of an evolutionarily conserved DOCK180-related superfamily of exchange factors (Cote, 2002).
Neutrophils are highly motile leukocytes, and they play important roles in the innate immune response to invading pathogens. Neutrophil chemotaxis requires Rac activation, yet the Rac activators functioning downstream of chemoattractant receptors remain to be determined. This study shows that DOCK2, a mammalian homologue of C. elegans CED-5 and Drosophila Myoblast City, regulates motility and polarity during neutrophil chemotaxis. Although DOCK2-deficient neutrophils move toward the chemoattractant source, they exhibit abnormal migratory behavior with a marked reduction in translocation speed. In DOCK2-deficient neutrophils, chemoattractant-induced activation of both Rac1 and Rac2 are severely impaired, resulting in the loss of polarized accumulation of F-actin and phosphatidylinositol 3,4,5-triphosphate (PIP3) at the leading edge. In contrast, that DOCK2 associates with PIP3 and translocates to the leading edge of chemotaxing neutrophils in a phosphatidylinositol 3-kinase-dependent manner. These results indicate that during neutrophil chemotaxis DOCK2 regulates leading edge formation through PIP3-dependent membrane translocation and Rac activation (Kunisaki, 2006; full text of article).
Myoblast fusion follows a defined sequence of events that is strikingly similar in vertebrates and invertebrates. Genetic analysis in Drosophila has identified many of the molecules that mediate the different steps in the fusion process; by contrast, the molecular basis of myoblast fusion during vertebrate embryogenesis remains poorly characterised. A key component of the intracellular fusion pathway in Drosophila is the protein encoded by the myoblast city (mbc) gene, a close homologue of the vertebrate protein dedicator of cytokinesis 1 (DOCK1, formerly DOCK180). Using morpholino antisense-oligonucleotide-mediated knockdown of gene activity in the zebrafish embryo, this study shows that the fusion of embryonic fast-twitch myoblasts requires the activities of Dock1 and the closely related Dock5 protein. In addition, it is shown that the adaptor proteins Crk and Crk-like (Crkl), with which Dock proteins are known to interact physically, are also required for myoblast fusion (Moore, 2007)
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