Isolation of a NCK-associated Kinase, PRK2, an SH3-binding Protein and Potential Effector of Rho Protein Signaling

Abstract

The NCK adapter protein is comprised of three consecutive Src homology 3 (SH3) protein-protein interaction domains and a C-terminal SH2 domain. Although the association of NCK with activated receptor proteintyrosine 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 we screened a bacterial expression library to isolate proteins that bind its 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 bound the SH3 domains of several signaling proteins, PRK2 specifically bound to the middle SH3 domain of NCK and (weakly) that of phospholipase C␥. PRK2 also specifically bound to Rho in a GTP-dependent manner and cooperated 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.Upon association with ligand, receptor protein-tyrosine kinases (PTKs) 1 dimerize and transphosphorylate on tyrosine residues. These phosphotyrosine-containing sequences then provide docking sites for SH2 and PTB domains, modular protein-protein interaction domains that bind to phosphotyrosine in the context of adjacent C-or N-terminal peptide sequences, respectively (1). In addition to recruiting signaling enzymes such as phosphoinositide 3-kinase, phospholipase C␥, and p120 GAP, adapter molecules including Grb2, Crk, and NCK also bind to PTKs via their SH2 domains. While lacking enzymatic activities, these adaptor proteins possess SH3 domains that bind to proline-rich sequences, minimally containing a consensus PXXP motif, in other signaling molecules (1-3). Possibly the best characterized adapter molecule is Grb2 that binds to PXXP motifs in the C terminus of the Ras guanine nucleotide exchange factor, Sos, recruiting it to the plasma membrane where it promotes Ras activation (4).The role of the adapter molecule NCK is less clearly understood. NCK is made up of three consecutive SH3 domains and a C-terminal SH2 domain (5). While the SH2 domain binds to a number of receptor-PTKs, including the epidermal, plateletderived, and vascular endothelial cell growth factor receptors as well as the insulin receptor substrate, IRS-1 (6 -8), little is known about the putative effector proteins that bind to its three SH3 domains. NCK's SH3 domains have been shown to bind to Cbl (9), a major PTK substrate and possible inhibitor of cellular signaling (10), as well as Sos1 (11), two molecules that also associate with Grb2 (12). However, unlike Grb2 (13), 2 NCK has also be...

Cytoskeletal Rearrangements and Transcriptional Activation of c-fos Serum Response Element by Rho-kinase

“…PRK2 shows strong sequence homology with protein kinase N (43) and is identified as a Rho target (44). PRK2 weakly potentiates the transcriptional activation of SRE by activated Rho (44). Consistently, the constitutively active form of protein kinase N weakly activates SRE (up to 2-fold) (data not shown).…”

“…PRK2 shows strong sequence homology with protein kinase N (43) and is identified as a Rho target (44). PRK2 weakly potentiates the transcriptional activation of SRE by activated Rho (44).…”

STAR syndrome-associated CDK10/Cyclin M regulates actin network architecture and ciliogenesis

“…39,40,[44][45][46][47] Our study identifies CDK10/CycM as a key upstream regulator of this biology. Combining our and previous observations, we propose a model in which CDK10/ CycM phosphorylates PKN2 within its RhoA binding domain, thereby promoting RhoA binding and stabilization, which results in actin polymerization and consequent repression of primary cilia formation at cell cycle exit (Fig.…”

Nck-2, a Novel Src Homology2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways

“…Lee et al, 1993;Chou and Hanafusa, 1995;Hu et al, 1995;Rivero-Lezcano et al, 1995;Birge et al, 1996;Bokoch et al, 1996;Galisteo et al, 1996;Kitamura et al, 1996;Quilliam et al, 1996;Lu et al, 1997;Lussier and Larose, 1997;Su et al, 1997;Anton et al, 1998). Nck is capable of physically associating with the signaling proteins through direct or indirect protein-protein interactions.…”