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

The Rho family GTPases Cdc42, Rac1 and RhoA regulate the reorganization of the actin cytoskeleton induced by extracellular signals such as growth factors. In mammalian cells, Cdc42 regulates the formation of filopodia, whereas Rac regulates lamellipodia formation and membrane ruffling, and RhoA regulates the formation of stress fibers. The serine/threonine protein kinase p65(pak) autophosphorylates, thereby increasing its catalytic activity toward exogenous substrates. This kinase is therefore a candidate effector for the changes in cell shape induced by growth factors. The microinjection of activated Pak1 protein into quiescent Swiss 3T3 cells induces the rapid formation of polarized filopodia and membrane ruffles. The prolonged overexpression of Pak1 amino-terminal mutants that are unable to bind Cdc42 or Rac1 results in the accumulation of filamentous actin in large, polarized membrane ruffles and the formation of vinculin-containing focal complexes within these structures. This phenotype resembles that seen in motile fibroblasts. The amino-terminal Pak1 mutant displays enhanced binding to the adaptor protein Nck, which contains three Src-homology 3 (SH3) domains. Mutation of a proline residue within a conserved SH3-binding region at the amino terminus of Pak1 interferes with SH3-protein binding and alters the effects of Pak1 on the cytoskeleton. These results indicate that Pak1, acting through a protein that contains an SH3 domain, regulates the structure of the actin cytoskeleton in mammalian cells, and may serve as an effector for Cdc42 and/or Rac1 in promoting cell motility (Sells, 1997).

Upon growth factor stimulation, Nck is recruited to receptor tyrosine kinases via its SH2 domain, probably initiating one or more signaling cascades. Nck is bound in living cells to the serine-threonine kinase Pak1. The association between Nck and Pak1 is mediated by the second SH3 domain of Nck and a proline-rich sequence in the amino terminus of Pak1. Pak1 is recruited by activated epidermal growth factor (EGF) and platelet-derived growth factor receptors. Moreover, Pak1 kinase activity is increased in response to EGF in HeLa cells transfected with human Pak1, and the kinase activity is enhanced when Nck is co-transfected. It is concluded that Nck links receptor tyrosine kinases with Pak1 and is probably involved in targeting and regulation of Pak1 activity (Galisteo, 1996).

The p21-activated kinases (PAKs) link G protein-coupled receptors and growth factor receptors to activation of MAP kinase cascades and to cytoskeletal reorganization. The proteins that interact with PAK to mediate its cellular effects and to couple it to upstream receptors are unknown. A specific interaction of the Nck adapter molecule with PAK1 both in vitro and in vivo is described. PAK1 and Nck associate in COS-7 and Swiss 3T3 cells constitutively, but this interaction is strengthened upon platelet-derived growth factor receptor stimulation. Nck binds to PAK1 through its second Src homology 3 (SH3) domain, while PAK1 interacts with Nck via the first proline-rich SH3 binding motif at its amino terminus. The interaction of active PAK1 with Nck leads to the phosphorylation of Nck at multiple sites. Association of Nck with PAK1 may serve to link this important regulatory kinase to cell activation by growth factor receptors (Bokoch, 1996).

The Pak family of serine/threonine kinases are known to be activated by binding to the GTP-bound form of Cdc42 or Rac1, which are small GTPases of the Rho family that are involved in regulating the organization of the actin cytoskeleton. Evidence is presented that Nck can mediate the relocalization and subsequent activation of the Pak1 kinases. Nck associates in vivo with Pak using the second of its three SH3 domains, and localization of this individual Nck SH3 domain, or of Pak kinase itself, to the membrane results in activation of Pak and stimulation of downstream mitogen activated protein kinase cascades. Activation of downstream signaling by the membrane-localized Nck SH3 domain is blocked by a kinase-inactive mutant form of Pak1. These results demonstrate that localization of Pak1 to the membrane in the absence of other signals is sufficient for its activation, and imply that the Nck adaptor protein could function to link changes in tyrosine phosphorylation of cellular proteins to the Cdc42/Pak signaling pathway (Lu, 1997).

Pak kinases are a family of serine/threonine protein kinases homologous to Ste20p of yeast. Paks can be activated in vivo and in vitro by binding to GTP-bound Cdc42 and Rac1, members of the Rho family of small GTPases implicated in regulating the organization of the actin cytoskeleton. The SH2/SH3-containing adaptor protein Nck binds Pak kinase through its second SH3 domain. Pak1 can be targeted to the membrane by Nck in response to tyrosine phosphorylation, and membrane association of Pak1 is sufficient to increase its specific activity. The mechanism whereby Pak is activated by membrane localization, however, is unknown. Expression of three proteins that inhibit Rho-family GTPases by different mechanisms (RhoGDI, Bcr and D57Y Cdc42) all block the activation of Pak by a membrane-targeted Nck SH3 domain, demonstrating that the in vivo activation of Pak1 induced by membrane localization is dependent on Rho-family GTPases. This implies that Pak activity can be regulated in cells both by the level of GTP loading of various Rho-family GTPases and the local concentration of Pak relative to these GTPases. These data also suggest the existence of Rho-family GTPases in addition to Cdc42 and Rac1 that can activate Pak on membranes (Lu, 1999).

Other downstream targets of Nck

NCK, an SH2- and SH3 domain-containing protein, becomes phosphorylated and associated with tyrosine kinase receptors upon growth factor stimulation. The sequence of NCK suggests that NCK functions as a linker between receptors and a downstream signaling molecule. To determine if NCK can mediate growth factor-stimulated responses, the ability of NCK to activate the fos promoter was measured. In NIH 3T3 cells, NCK strongly activates this promoter. The effect of NCK on the fos promoter is enhanced by c-ras and blocked by dominant negative ras. NCK binds directly to the guanine nucleotide exchange factor SOS. This interaction is mediated by the SH3 domains of NCK. These findings suggest that NCK can regulate p21ras-dependent gene transcription through interaction with SOS protein (Hu, 1995).

A novel mammalian serine/threonine kinase that interacts with the SH3 domains of Nck has been identified and termed Nck Interacting Kinase (NIK). This kinase is most homologous to the Sterile 20 (Ste20) family of protein kinases. Of the members of this family, GCK and MSST1 are most similar to NIK in that they bind neither Cdc42 nor Rac and contain an N-terminal kinase domain with a putative C-terminal regulatory domain. Transient overexpression of NIK specifically activates the stress-activated protein kinase (SAPK) pathway. Both the kinase domain and C-terminal regulatory region of NIK are required for full activation of SAPK. NIK likely functions upstream of MEKK1 to activate this pathway; a dominant-negative MEK kinase 1 (MEKK1) blocks activation of SAPK by NIK. MEKK1 and NIK also associate in cells and this interaction is mediated by regulatory domains on both proteins. Two other members of this kinase family, GCK and HPK1, contain C-terminal regulatory domains with homology to the C-terminal domain NIK. These findings indicate that the C-terminal domains of these proteins encode a new protein domain family and suggests that this domain couples these kinases to the SAPK pathway, possibly by interacting with MEKK1 or related kinases (Su, 1997).

Nck is a 47-kDa cytosolic protein devoid of intrinsic catalytic activity and consisting of Src homology 2 and 3 (SH2 and SH3) domains organized as follows: SH3-SH3-SH3-SH2. Nck is believed to act as an adaptor protein mediating signal transduction initiated by receptor tyrosine kinases (RTKs). Through its SH2 domain, Nck recognizes a specific phosphotyrosine residue on RTKs or on protein substrates of RTKs like insulin receptor substrate-1, the major substrate of the insulin receptor. Through its SH3 domains it interacts with poorly characterized effector molecules. To identify novel proteins that might interact with Nck, the amino-terminal segment of Nck encompassing its three SH3 domains was used in the yeast two-hybrid system. Among the polypeptides that associate with Nck, the gamma2 isoform of the serine/threonine casein kinase I (CKI-gamma2) was identified. In transformed rat hepatocytes overexpressing the insulin receptor (HTC-IR cells), serine/threonine protein kinase activity coimmunoprecipitates with Nck, an interaction mediated mainly by the third SH3 domain of Nck. This kinase activity is not apparently modulated by insulin, nor is it sensitive to staurosporine or heparin, and it does not use GTP as a phosphate donor. However the kinase activity coimmunoprecipitated with Nck is completely abolished by a specific inhibitor of casein kinase I. In an in vitro renaturation gel kinase assay, a protein kinase of 70-75 kDa has been detected associated with the SH3 domains of Nck. The SH3 domains of Nck bind directly to a cytosolic protein of 70-75 kDa. A rabbit polyclonal antibody raised against the C-terminal region of CKI-gamma2 protein kinase immunoprecipitates a single specific protein of 70-75 kDa from HTC-IR cell lysates and detects CKI-gamma2 among the proteins coimmunoprecipitated with Nck. These results support an in vivo interaction between Nck and CKI-gamma2 and suggest that CKI-gamma2 could be involved in signaling pathways downstream of RTKs (Lussier, 1997).

Dok, a 62-kDa Ras GTPase-activating protein (rasGAP)-associated phosphotyrosyl protein, is thought to act as a multiple docking protein downstream of receptor or non-receptor tyrosine kinases. Cell adhesion to extracellular matrix proteins induces marked tyrosine phosphorylation of Dok. This adhesion-dependent phosphorylation of Dok is mediated, at least in part, by Src family tyrosine kinases. The maximal insulin-induced tyrosine phosphorylation of Dok requires a Src family kinase. A mutant Dok (DokdeltaPH) that lacks its pleckstrin homology domain failed to undergo tyrosine phosphorylation in response to cell adhesion or insulin. Furthermore, unlike the wild-type protein, DokdeltaPH does not localize to subcellular membrane components. Insulin promotes the association of tyrosine-phosphorylated Dok with the adapter protein NCK and rasGAP. In contrast, a mutant Dok (DokY361F), in which Tyr361 is replaced by phenylalanine, fails to bind NCK but partially retains the ability to bind rasGAP in response to insulin. Overexpression of wild-type Dok, but not that of DokdeltaPH or DokY361F, enhances the cell migratory response to insulin without affecting insulin activation of mitogen-activated protein kinase. These results identify Dok as a signal transducer that potentially links, through its interaction with NCK or rasGAP, cell adhesion and insulin receptors to the machinery that controls cell motility (Noguchi, 1999).

Nck functions in the nucleus

Confocal microscopy was used to localize Nck in NIH3T3 and A431 cells. Surprisingly, Nck has been identified in the nucleus as well as the cytoplasm with no visible change in localization due to PDGF or EGF stimulation. Western blot analysis of nuclear and cytosolic fractions confirms that there is no translocation in response to growth factor and that tyrosine phosphorylation is specific to only cytosolic Nck. Far Western blot analysis with either Nck, the SH2 domain, or the SH3 domains reveal differential binding in nuclear and cytosolic lysates, indicating specific binding partners for each subcellular location. The major target of c-Src during mitosis is SAM68, a RNA-binding protein ordinarily localized to the nucleus. SAM68 was identified as a nuclear specific binding partner of Nck in both nonmitotic and mitotic cells. Several tyrosine kinases can be found in the nucleus but their signal transduction remains undefined. The discovery of an adaptor protein in the nucleus suggests there are signal transduction mechanisms within the nucleus that recapitulate those found in the cytoplasm (Lawe, 1997).

Nck and the immune response

The T-cell antigen receptor (TCR) triggers a signaling cascade initiated by the tyrosine kinase Lck and requiring the proto-oncogene p95(vav). Vav is activated by Lck and can function as a guanine nucleotide exchange factor for the Rho-family GTPases, Rac1 and Cdc42. To investigate the involvement of these GTPases in TCR signaling, their well characterized effector, Pak1, was studied. This serine/threonine kinase is activated by GTP-bound Rac1 or Cdc42. However, its role in mediating downstream signaling events is controversial. Rapid, TCR-dependent activation of Pak1 is observed and also TCR-inducible association of Pak1 with Nck, which is tyrosine phosphorylated following stimulation. Pak1 activation occurs independent of Ras activation or calcium flux, but is dependent on the Lck tyrosine kinase, and is downstream of Vav and Cdc42. Dominant negative Pak1 or Nck specifically inhibits TCR-mediated activation of the nuclear factor of activated T cells (NFAT) transcription factor. TCR-mediated activation of Erk2 is also inhibited by dominant negative Pak. However, Pak1 activation is neither necessary nor sufficient for TCR-dependent c-Jun N-terminal kinase (JNK) activation. Therefore, Pak1 acts downstream of Vav and is required for activation of Erk2 and NFAT by a JNK-independent pathway. This is the first demonstration of a requirement for Pak to mediate the regulation of gene expression by an extracellular ligand (Yablonski, 1998)

Nck and development

Nck is a widely expressed SH2/SH3 adaptor protein containing one SH2 and three SH3 domains. Although Nck is assumed to mediate the formation of protein-protein complexes during signaling, little is currently known about its specific function. A series of Nck SH3 and SH2 domain mutants were constructed; they were expressed in Xenopus laevis embryos, and injected embryos were monitored for developmental abnormalities. This approach allows correlation of developmental phenotypes with the presence or absence of specific Nck protein-binding domains. Microinjection of RNA-encoding Nck with an inactivating mutation in the third SH3 domain (NckK229) into dorsal blastomeres of early embryos causes anterior truncation with high frequency, and membrane localization of both the first and second SH3 domains together is sufficient to induce this anterior-truncation phenotype. Molecular marker analysis of explants reveals that the expression of NckK229 ventralizes dorsal mesoderm. Lineage tracing experiments demonstrates that the expression of Nck K229 in dorsal blastomeres affects the migratory properties of mesoderm cells in gastrulation and leads to the adoption of a more posterior fate. These data suggest that protein(s) that bind the first and second SH3 domains of Nck can affect the response to signals that establish dorso-ventral patterning, and that protein(s) that binds the third SH3 domain antagonizes the ventralizing effect of the first two SH3 domains (Tanaka, 1997).

Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. The murine Nck1 and Nck2 genes were mutated and incorporated beta-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1(-/-) Nck2(-/-) embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization (Bladt, 2003).

The glomerular filtration barrier in the kidney is formed in part by a specialized intercellular junction known as the slit diaphragm, which connects adjacent actin-based foot processes of kidney epithelial cells (podocytes). Mutations affecting a number of slit diaphragm proteins, including nephrin (encoded by NPHS1), lead to renal disease owing to disruption of the filtration barrier and rearrangement of the actin cytoskeleton, although the molecular basis for this is unclear. This study shows that nephrin selectively binds the Src homology 2 (SH2)/SH3 domain-containing Nck adaptor proteins, which in turn control the podocyte cytoskeleton in vivo. The cytoplasmic tail of nephrin has multiple YDxV sites that form preferred binding motifs for the Nck SH2 domain once phosphorylated by Src-family kinases. This Nck-nephrin interaction is required for nephrin-dependent actin reorganization. Selective deletion of Nck from podocytes of transgenic mice results in defects in the formation of foot processes and in congenital nephrotic syndrome. Together, these findings identify a physiological signalling pathway in which nephrin is linked through phosphotyrosine-based interactions to Nck adaptors, and thus to the underlying actin cytoskeleton in podocytes. Simple and widely expressed SH2/SH3 adaptor proteins can therefore direct the formation of a specialized cellular morphology in vivo (Jones, 2006).

Nck's role as an oncogene

Human nck has been established as a new oncogene. nck encodes one SH2 and three SH3 domains; the Src homology motifs found in nonreceptor tyrosine kinases; Ras GTPase-activating protein; phosphatidylinositol 3-kinase, and phospholipase C-gamma. Overexpression of human nck in 3Y1 rat fibroblasts results in transformation as judged by alteration of cell morphology, colony formation in soft agar, and tumor formation in nude BALB/c mice. However, overexpression of nck does not induce detectable elevation of the phosphotyrosine content of specific proteins, as is observed for v-crk, another SH2/SH3-containing oncogene. Despite this fact, Nck retains the ability to bind tyrosine phosphorylated proteins in vitro, using a fusion protein of Nck with glutathione-S-transferase (GST). Moreover, when incubated with lysates prepared from v-src-transformed 3Y1 cells or the nck-overexpressing cell lines, GST-Nck binds to both p60v-src and serine/threonine kinases, respectively. Although phosphotyrosine levels are not elevated in the nck-expressing fibroblasts, vanadate treatment of these cells results in a phosphotyrosine pattern that is altered from the parental 3Y1 pattern, suggestive of a perturbation of indigenous tyrosine kinase pathways. These results suggest the possibility that human nck induces transformation in 3Y1 fibroblasts by virtue of its altered affinity or specificity for the normal substrates of its rat homolog and that Nck may play a role in linking tyrosine and serine/threonine kinase pathways within the cell (Chou, 1992).

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

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