FAK functions upstream of NCK

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes the Src homology 2 (SH2) domain binding of the Src family PTKs; c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. FN-stimulated signaling to ERK2 was examined and it was found that ERK2 activation is reduced 10-fold in Src- fibroblasts, as compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK are reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas (see CAS/CSE1 segregation protein) , is not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocks Grb2 binding to FAK, but surprisingly, Src 1-298 expression also results in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity, when compared to levels in Src- cells. Src 1-298 bind to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK phosphorylates p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promotes the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK (Schlaepfer,1997).

Tek/Tie2 is an endothelial cell-specific receptor tyrosine kinase that has been shown to play a role in vascular development of the mouse. Targeted mutagenesis of both Tek and its agonistic ligand, Angiopoietin-1, result in embryonic lethality, demonstrating that the signal transduction pathway(s) mediated by this receptor are crucial for normal embryonic development. In an attempt to identify downstream signaling partners of the Tek receptor, the yeast two-hybrid system was used to identify phosphotyrosine-dependent interactions. Using this approach, a novel docking molecule called Dok-R was identified, which has sequence and structural homology to p62dok and IRS-3. Mapping of the phosphotyrosine-interaction domain within Dok-R shows that Dok-R interacts with Tek through a PTB domain. Dok-R is coexpressed with Tek in a number of endothelial cell lines. Coexpression of Dok-R with activated Tek results in tyrosine phosphorylation of Dok-R and rasGAP and Nck coimmunoprecipitate with phosphorylated Dok-R. Furthermore, Dok-R is constitutively bound to Crk presumably through the proline rich tail of Dok-R. The cloning of Dok-R represents the first downstream substrate of the activated Tek receptor, and suggests that Tek can signal through a multitude of pathways (Jones, 1998).

Pak functions upstream of Nck

The p21-activated kinases (PAKs) are important mediators of cytoskeletal reorganization, cell motility and transcriptional events regulated by the Rho family GTPases Rac and Cdc42. PAK activation by serum components is strongly dependent on cell adhesion to the extracellular matrix (ECM). PAK binds directly to the Nck adapter protein, an interaction thought to play an important role in regulation and localization of PAK activity. The interaction of PAK with Nck is regulated dynamically by cell adhesion. PAK-Nck binding is rapidly lost after cell detachment and rapidly restored after re-adhesion to the ECM protein fibronectin, suggesting a rapidly reversible mode of regulation. Furthermore, the loss of Nck binding correlates with changes in the phosphorylation state of PAK in nonadherent cells, as evidenced by electrophoretic mobility shift and phosphorylation within a sequence known to mediate interaction with Nck. The ability of cell adhesion to regulate PAK phosphorylation and interaction with Nck may contribute to the anchorage-dependence of PAK activation as well as to the localization of activated PAK within a cell.

Septins regulate actin organization and cell-cycle arrest through nuclear accumulation of NCK mediated by SOCS7

Mammalian septins are GTP-binding proteins the functions of which are not well understood. Knockdown of SEPT2, 6, and 7 causes stress fibers to disintegrate and cells to lose polarity. This phenotype is induced by nuclear accumulation of the adaptor protein NCK; the effects can be reversed or induced by cytoplasmic or nuclear NCK, respectively. NCK is carried into the nucleus by SOCS7 (suppressor of cytokine signaling 7), which possesses nuclear import/export signals. SOCS7 interacts with septins and NCK through distinct domains. DNA damage induces actin and septin rearrangement and rapid nuclear accumulation of NCK and SOCS7. Moreover, NCK expression is essential for cell-cycle arrest. The septin-SOCS7-NCK axis intersects with the canonical DNA damage cascade downstream of ATM/ATR and is essential for p53 Ser15 phosphorylation. These data illuminate an unanticipated connection between septins, SOCS7, NCK signaling, and the DNA damage response (Kremer, 2007).

Nap1 connects Rac with Nck

Bacterially expressed glutathione S-transferase fusion proteins containing Rac1 were used to identify binding proteins of this Rho family GTPase present in a bovine brain extract. Five proteins of 85, 110, 125, 140 and 170 kDa were detected, all of which were associated exclusively with guanosine 5'-[gamma-thio]triphosphate-bound Rac1, not with GDP-bound Rac1. The 85 and 110 kDa proteins were identified as the regulatory and catalytic subunits respectively of phosphatidylinositol 3-kinase. Several lines of evidence suggested that the 125 kDa protein is identical with Nck-associated protein 1 (Nap1). The mobilities of the 125 kDa protein and Nap1 on SDS/PAGE were indistinguishable, and the 125 kDa protein was depleted from brain extract by preincubation with the Src homology 3 domain of Nck to which Nap1 binds. Furthermore, antibodies to Nap1 reacted with the 125 kDa protein. Nap1 was co-immunoprecipitated with a constitutively active form of Rac expressed in Chinese hamster ovary cells. The observation that complex formation between activated Rac and PAK, but not that between Rac and Nap1, could be reproduced in vitro with recombinant proteins indicates that the interaction of Nap1 with Rac is indirect. The 140 kDa Rac-binding protein is a potential candidate for a link that connects Nap1 to Rac. The multimolecular complex comprising Rac, Nap1 and probably the 140 kDa protein might mediate some of the biological effects transmitted by the multipotent GTPase (Kitamura, 1997).

SOCS3 interacts with the Nck and Crk-L adapter proteins and regulates Nck activation

Suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor signal transduction. Although the affect of SOCS proteins on the Jak-STAT pathway has been well characterized, their role in the regulation of other signaling modules is not well understood. In the present study, it has been demonstrated that SOCS3 physically interacts with the SH2/SH3-containing adapter proteins Nck and Crk-L, which are known to couple activated receptors to multiple downstream signaling pathways and the actin cytoskeleton. SOCS3/Nck and SOCS3/Crk-L interactions depend on tyrosine phosphorylation of SOCS3 Tyr(221) within the conserved SOCS box motif and intact SH2 domains of Nck and Crk-L. Furthermore, SOCS3 Tyr(221) forms a YXXP motif, which is a consensus binding site for the Nck and Crk-L SH2 domains. Expression of SOCS3 in NIH3T3 cells induces constitutive recruitment of a Nck-GFP fusion protein to the plasma membrane and constitutive tyrosine phosphorylation of endogenous Nck. These findings suggest that SOCS3 regulates multiple cytokine and growth factor-activated signaling pathways by acting as a recruitment factor for adapter proteins (Sitko, 2004).

WASP functions downstream of NCK

In the second of a series of experiments designed to identify p47nck-Src homology 3 (SH3)-binding molecules, the cloning of SAKAP II (Src A box Nck-associated protein II) from an HL60 cDNA expression library is described. This molecule has been identified as a cDNA encoding the protein product of WASP, which is mutated in Wiskott-Aldrich syndrome patients. Studies in vivo and in vitro demonstrate a highly specific interaction between the SH3 domains of p47nck and Wiskott-Aldrich syndrome protein. Furthermore, anti-Wiskott-Aldrich syndrome protein antibodies recognize a protein of 66 kDa by Western blot (immunoblot) analysis. In vitro translation studies have identified the 66-kDa protein as the protein product of WASP, and subcellular fractionation experiments show that p66WASP is mainly present in the cytosol fraction, although significant amounts are also present in membrane and nuclear fractions. The main p47nck region implicated in the association with p66WASP is the carboxy-terminal SH3 domain (Rivero-Lezcano, 1995).

Although the association of NCK with activated receptor protein-tyrosine kinases, via its SH2 domain, implicates NCK as a mediator of growth factor-induced signal transduction, little is known about the pathway(s) downstream of NCK recruitment. To identify potential downstream effectors of NCK a bacterial expression library was screened to isolate proteins that bind NCK's SH3 domains. Two molecules were isolated: the Wiskott-Aldrich syndrome protein (WASp, a putative CDC42 effector) and a serine/threonine protein kinase (PRK2, closely related to the putative Rho effector PKN). Using interspecific backcross analysis, the Prk2 gene was mapped to mouse chromosome 3. Unlike WASp, which binds the SH3 domains of several signaling proteins, PRK2 specifically binds to the middle SH3 domain of NCK and (weakly) binds to the phospholipase Cgamma. PRK2 also specifically bind to Rho in a GTP-dependent manner and cooperates with Rho family proteins to induce transcriptional activation via the serum response factor. These data suggest that PRK2 may coordinately mediate signal transduction from activated receptor protein-tyrosine kinases and Rho and that NCK may function as an adapter to connect receptor-mediated events to Rho protein signaling (Quilliam, 1996).

Nck is a ubiquitous adaptor molecule composed of three Src homology 3 (SH3) domains followed by a single SH2 domain. Nck links, via its SH2 domain, tyrosine-phosphorylated receptors to effector proteins that contain SH3-binding proline-rich sequences. Recombinant Nck precipitates endogenous WIP, a novel proline-rich protein that interacts with the Wiskott-Aldrich syndrome protein (WASp), from BJAB cell lysates. Nck binds through its second SH3 domain to WIP, and Nck binds to WIP at a site (amino acids 321-415) that differs from the WASp-binding site (amino acids 416-488). WIP has been shown to associate with the actin polymerization regulatory protein profilin and to induce actin polymerization and cytoskeletal reorganization in lymphoid cells. The presence of profilin in Nck precipitates is demonstrated, suggesting that Nck may couple extracellular signals to the cytoskeleton via its interaction with WIP and profilin (Anton, 1997).

Src homology domains [i.e., Src homology domain 2 (SH2) and Src homology domain 3 (SH3)] play a critical role in linking receptor tyrosine kinases to downstream signaling networks. A well-defined function of the SH3-SH2-SH3 adapter Grb2 is to link receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), to the p21ras-signaling pathway. Grb2 has also been implicated as having a role in growth factor-regulated actin assembly and receptor endocytosis, although the underlying mechanisms remain unclear. Grb2 interacts through its SH3 domains with the human Wiskott-Aldrich syndrome protein (WASp), which plays a role in regulation of the actin cytoskeleton. WASp is expressed in a variety of cell types and is exclusively cytoplasmic. Although the N-terminal SH3 domain of Grb2 binds to WASp with significantly more strength than the C-terminal SH3 domain binds to WASp, full-length Grb2 shows the strongest binding. Both phosphorylation of WASp and its interaction with Grb2, as well as with another adapter protein (Nck), remain constitutive in serum-starved or epidermal growth factor-stimulated cells. WASp coimmunoprecipitates with the activated EGFR after epidermal growth factor stimulation. Purified glutathione S-transferase-full-length-Grb2 fusion protein, but not the individual domains of Grb2, enhances the association of WASp with the EGFR, suggesting that Grb2 mediates the association of WASp with EGFR. This study suggests that Grb2 translocates WASp from the cytoplasm to the plasma membrane and the Grb2-WASp complex may play a role in linking receptor tyrosine kinases to the actin cytoskeleton (She, 1997).

Wiskott-Aldrich syndrome protein (WASP) activation at the site of T cell-APC interaction is a two-step process, with recruitment dependent on the proline-rich domain and activation dependent on binding of Cdc42-GTP to the GTPase binding domain. WASP recruitment occurs through binding to the C-terminal Src homology 3 domain of Nck. In contrast, WASP activation requires Vav-1. In Vav-1-deficient T cells, WASP recruitment proceeds normally, but localized activation of Cdc42 and WASP is disrupted. The recruitment and activation of WASP are coordinated by tyrosine-phosphorylated Src homology 2 domain-containing leukocyte protein of 76 kDa, which functions as a scaffold, bringing Nck and WASP into proximity with Vav-1 and Cdc42-GTP. Taken together, these findings reconstruct the signaling pathway leading from TCR ligation to localized WASP activation (Zeng, 2003).

Nck acts upstream of Sos and dynamin

One of the adaptor proteins, Nck, comprises a single SH2 domain and three SH3 domains that are important in protein-protein interactions. The in vivo association of Nck with the guanine nucleotide exchange factor Sos has been well documented; however, the precise nature of the interaction is unclear. To determine which SH3 domains are involved in the Nck-Sos interaction, individual SH3 domains of Nck were generated as glutathione S-transferase fusion proteins. Exclusively the third (C-terminal) SH3 domain of Nck has the ability to bind to Sos. In addition, in [35S]methionine labelled K562 cells, a 100,000 Mr protein is found to be associated with the third SH3 domain of Nck. This protein has been identified as dynamin, a GTP-binding protein that has been implicated in clathrin-coated vesicle formation. Dynamin and Nck co-precipitate when cell lysates are immunoprecipitated with anti-Nck antibody. These data suggest that Nck may contribute to Ras activation and the function of dynamin in membrane trafficking through its third SH3 domain (Wunderlich, 1999).

p125Nap1 associates with an SH3 domain of Nck

Binding proteins to the Src homology 3 (SH3) domains of Nck were screened by the use of glutathione S-transferase fusion proteins. Two proteins of 140 and 125 kDa were detected, both of which associated preferentially with the first SH3 domain of Nck. The 125-kDa protein, designated as Nap1 for Nck-associated protein 1, was purified and the corresponding rat cDNA was isolated. The predicted amino acid sequence revealed that p125Nap1 does not contain any known functional motif but shows sequence homology to Hem family gene. Using specific antibodies, p125Nap1 was shown to associate with Nck both in vitro and in intact cells. Further characterization of p125Nap1 may clarify the protein-protein interaction in the downstream signaling of Nck (Kitamura, 1996).

Nck interacts with PINCH-1

Weak protein-protein interactions (PPIs) are critical determinants of many biological processes. However, in contrast to a large growing number of well-characterized, strong PPIs, the weak PPIs are poorly explored. Genome wide, there exist few 3D structures of weak PPIs with, and none with. This study reports the NMR structure of an extremely weak focal adhesion complex between Nck-2 SH3 domain and PINCH-1 LIM4 domain (see Steamer duck). The structure exhibits a remarkably small and polar interface with distinct binding modes for both SH3 and LIM domains. Such an interface suggests a transient Nck-2/PINCH-1 association process that may trigger rapid focal adhesion turnover during integrin signaling. Genetic rescue experiments demonstrate that this interface is indeed involved in mediating cell shape change and migration. Together, the data provide a molecular basis for an ultraweak PPI in regulating focal adhesion dynamics during integrin signaling (Vaynberg, 2005).

PINCH-1, a widely expressed protein consisting of five LIM domains and a C-terminal tail, is an essential focal adhesion protein with multiple functions including regulation of the integrin-linked kinase (ILK) level, cell shape, and survival signaling. The LIM1-mediated interaction with ILK regulates all these three processes. By contrast, the LIM4-mediated interaction with Nck-2, which regulates cell morphology and migration, is not required for the control of the ILK level and survival. Remarkably, a short 15-residue tail C-terminal to LIM5 is required for both cell shape modulation and survival, albeit it is not required for the control of the ILK level. The C-terminal tail not only regulates PINCH-1 localization to focal adhesions but also functions after it localizes there. These findings suggest that PINCH-1 functions as a molecular platform for coupling and uncoupling diverse cellular processes via overlapping but yet distinct domain interactions (Xu, 2005).

Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck

Rac signalling to actin -- a pathway that is thought to be mediated by the protein Scar/WAVE (WASP (Wiskott-Aldrich syndrome protein: see Drosophila Scar)-family verprolin homologous protein) -- has a principal role in cell motility. In an analogous pathway, direct interaction of Cdc42 with the related protein N-WASP stimulates actin polymerization. For the Rac-WAVE pathway, no such direct interaction has been identified. This study reports a mechanism by which Rac and the adapter protein Nck activate actin nucleation through WAVE1. WAVE1 exists in a heterotetrameric complex that includes orthologues of human PIR121 [p53-inducible messenger RNA with a relative molecular mass (Mr) of 140,000], Nap125 (NCK-associated protein with an Mr of 125,000) and HSPC300. Whereas recombinant WAVE1 is constitutively active, the WAVE1 complex is inactive. It is therefore proposed that Rac1 and Nck cause dissociation of the WAVE1 complex, which releases active WAVE1-HSPC300 and leads to actin nucleation (Eden, 2002).

Members of the Rho family of small GTPases, such as Cdc42 and Rac1, and of the Src homology (SH) domain-containing SH2–SH3 adapter protein family, such as NCK, link extracellular signals and actin nucleation through pathways that include the WASP family of proteins and the actin nucleation machinery—the Arp2/3 complex. All WASP family members contain a conserved verprolin-homology, cofilin-homology, acidic (VCA) domain that directly binds and activates the Arp2/3 complex. The Arp2/3 complex, in turn, catalyses the nucleation of actin filaments. To prevent undesirable spontaneous actin nucleation in the absence of input signals, the activity of the WASP proteins is tightly regulated. For example, N-WASP is found predominantly in an autoinhibited conformation in which the carboxy-terminal VCA domain is occluded through interaction with the amino terminus of the protein. When Cdc42 binds to the Cdc42/Rac1 interactive binding (CRIB) domain of N-WASP or when NCK binds to the polyproline region of N-WASP, this autoinhibition is relieved and the VCA domain is unmasked. Phosphatidylinositol(4,5)bisphosphate (PIP2) can further activate N-WASP in cooperation with NCK or Cdc42 by binding to a basic region of N-WASP (Eden, 2002).

The WAVE proteins (WAVE1, WAVE2 and WAVE3 in mammals and orthologues in Drosophila and Dictyostelium are similar in structure to N-WASP. They all have a C-terminal VCA domain, a polyproline region and a basic region. Unlike N-WASP, WAVE proteins do not contain a CRIB domain, and direct binding of WAVE1 to Rac1 has not been detected. But much evidence suggests that WAVE1 functions downstream of Rac1. WAVE1 is translocated from the cytoplasm to membrane ruffles induced by Rac1, and dominant-negative WAVE1 abolishes the formation of Rac1-dependent lamellipodia and Rac1-dependent neurite extensions. The mechanism of regulation of WAVE1 is likely to be fundamentally different from that of N-WASP: whereas N-WASP is autoinhibited, recombinant WAVE1 is constitutively active in stimulating the actin nucleation activity of Arp2/3. Therefore, WAVE1 activity is either inhibited in trans by other cellular regulators or regulated by post-translational modifications (Eden, 2002).

Although WAVE1 had been implicated as the downstream target of Rac1, no regulatory linkage had been found previously. Consequently, the important Rac1-dependent pathway for actin nucleation has not been described. Similarly, although the association of NCK and Rac1 with NAP125 and PIR121 has been observed in several screens, their role as regulators of actin nucleation has not been shown. These results indicate that WAVE1, like N-WASP and WASP, mediates signals from NCK and the Rho GTPases. The activation mechanisms of WAVE1 and N-WASP are very different: N-WASP is autoinhibited, whereas WAVE1 is trans-inhibited. The action of Rac1 and NCK is to disassemble the trans-inhibited WAVE1 complex, which releases the active WAVE1 protein in association with HSPC300. Consistent with this model, Rac1 and WAVE1 do not colocalize in the lamellipodium: WAVE1 is localized at the extreme edge of the lamellipodium, whereas Rac1 is distributed diffusely over the lamellipodium (Eden, 2002).

Although the predominant regulation of WAVE1 activity described in this study is relief of trans-inhibition, an additional positive regulation by proteins that bind WAVE1 directly in an activator-independent manner is also possible. For example, IRSp53 has been reported to bind WAVE2 directly and enhance activation of Arp2/3 by recombinant WAVE2. Preliminary data show that HSPC300, which remains associated with WAVE1 after activation, may also have a stimulating function on actin polymerization. The activation and dissociation of the WAVE1 complex process releases a subcomplex of NAP125 and PIR121, and this subcomplex may be free to interact with other cellular components. In this way, a Rac1 or NCK signal might potentially coordinate several cellular processes -- similar to pathways that are activated by the alpha- and the gamma-subunits in heterotrimeric G-protein signalling (Eden, 2002).

WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Tandem mass spectrometry has been used to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2-Abi1-Nap1-PIR121 complex. The WAVE2-Abi1-Nap1-PIR121 complex is as active as the WAVE2-Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo (Innocenti, 2004).

Pak1, a kinase activated by Rho family GTPases, also functions downstream of Nck

Evolutionary homologs continue: part 3/3 | back to part 1/3

dreadlocks: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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