Binding of NCK to SOS and Activation of ras-Dependent Gene Expression

Abstract

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 factorstimulated responses, we measured the ability of NCK to activate the fos promoter. We found that 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. We also found that 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 p21 ras -dependent gene transcription through interaction with SOS protein.NCK, a 45-kDa protein consisting of one SH2 domain and three SH3 domains, is expressed in a wide range of tissues and cell lines (20). After stimulation of cells by growth factors, NCK becomes phosphorylated on Ser, Thr, and Tyr residues and associates with tyrosine kinase receptors (5,17,21,25,28). In this way, NCK resembles other proteins that have SH2 domains and associate with receptors through interaction between SH2 domains and specific phosphotyrosines (13,19,24,29). Overexpression of NCK transforms mammalian fibroblasts, suggesting that its SH2 and SH3 domains may play important roles in cell growth regulation (5, 21). However, the mechanism of action of NCK is not understood.Recent studies of SH2-and SH3 domain-containing signaling molecules that associate with tyrosine-phosphorylated receptors have shown that signaling molecules are of two general types. The first type, typified by phospholipase C-␥, is an enzyme itself. The p85 subunit of phosphatidylinositol 3-kinase (10, 27, 34) represents a second type of receptor-associated molecule, in that it probably has no defined intrinsic enzyme activity but serves as a linker that connects an enzyme, in this case the catalytic subunit of phosphatidylinositol 3-kinase, to a receptor. Another example of the second type of linking molecule is the Sem5 gene product in Caenorhabditis elegans and its mammalian counterpart, GRB2. The SH2 domain of Sem5/ GRB2 binds to tyrosine kinase receptors (22,26,33). The SH3 region of Sem5/GRB2, like the SH3 region of the Abelson protein (6, 7, 30), binds to a proline-rich motif that is a common feature of a number of molecules implicated in growth regulation (9,16,26, 32,33).The absence of a recognizable catalytic domain of NCK suggests that NCK serves as a linker molecule connecting tyrosine-phosphorylated proteins to enzymes which might lack their own SH2 domains. It has been shown that NCK forms a physical complex with the platelet-derived growth factor (PDGF) receptor and the epidermal growth factor receptor in a ligand-dependent manner (17, 21). To determine if NCK can mediate growth factor-stimulated early cellular responses, we tested the ability of NCK to activate transcription of the fos gene, a well-characterized...

Mechanism of activation of Pak1 kinase by membrane localization

“…It has been reported that Nck can regulate p21ras-dependent gene transcription through interaction with SOS, an activator of Ras nucleotide exchange (Hu et al, 1995). This raised the possibility that activation of Pak1 via myristoylated Nck SH3(2) is an indirect e ect of activation of the Ras/Raf pathway due to membrane relocalization of SOS.…”

Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor

“…In these cells, Nck did not, however, interfere with the EGFand the NGF-stimulated Ras and MAPK activation (Rockow et al, 1996). On the other hand, it has also been reported that overexpression of Nck activated the c-fos promoter in a Ras-dependent manner in NIH3T3 cells, in which a stable complex of Nck and mSos was detected (Hu et al, 1995). The reason for this discrepancy among the above independent studies remains unknown.…”

Dominant-negative mutants of the SH2/SH3 adapters Nck and Grb2 inhibit MAP kinase activation and mesoderm-specific gene induction by eFGF in Xenopus

“…It has been proposed that the binding of Nck to p130 cas upon stimulation of Src 7 cells with ®bronectin might facilitate signaling to Erk2 (Schlaepfer et al, 1997). It has also been demonstrated that Nck can bind to Sos in vitro (Hu et al, 1995) potentially activating the Ras/Raf/MAPK pathway. On the other hand, PC12 cell di erentiation induced by basic FGF has been shown to be inhibited by Nck in a MAPK-independent mechanism (Rockow et al, 1996).…”

“…The three SH3 domains of Nck can bind to di erent downstream signaling proteins (Chou and Hanafusa, 1995;Hu et al, 1995;Kitamura et al, 1996;Lu et al, 1997;Quilliam et al, 1996;Rivero-Lezcano et al, 1994, 1995. We have previously shown that ectopic expression of Nck constructs in which both the ®rst and second SH3 domains are intact leads to ventralization and anterior truncation of Xenopus embryos (Tanaka et al, 1997).…”

“…Activated Ras interacts with Raf, a serine/ threonine kinase that phosphorylates and activates MAPK kinase (MEK) (Dent et al, 1992;Hughes et al, 1993;Kyriakis et al, 1992;MacDonald et al, 1993), which in turn phosphorylates Erk1 and Erk2 (MAPK) on threonine and tyrosine residues (Gomez and Cohen, 1991;Her et al, 1993;L'Allemain et al, 1992;Nakeilny et al, 1992;Rossomando et al, 1991). Although Nck and Crk have also been reported to bind to Sos (Feller et al, 1995;Hu et al, 1995;Okada and Pessin, 1996;Teng et al, 1995) and might therefore also link activated tyrosine kinases to the Ras/Raf/MAPK signaling pathway, little is known about the relevance of this binding in vivo. Nck and Crk can also propagate signals through other mechanisms.…”