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

doi:10.1074/jbc.271.46.28772

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

From the ^‡ Department of Biochemistry and Molecular Biology and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202, Departments of
^¶ Pharmacology and
** Biology and the
^‡‡ Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, and
^§§ Mammalian Genetics Laboratory, ABL Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702

^§To whom correspondence should be addressed:
Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr. MS 410, Indianapolis, IN 46202-5122.
Tel.: 317-274-8550; Fax: 317-274-4686; E-mail: lawrence_quilliam{at}iucc.iupui.edu.

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 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 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 Cγ. 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.

Footnotes

↵^∥ Supported by a Lineberger Cancer Center fellowship.
↵* This work was supported by National Cancer Institute Grants CA63139 (to L. A. Q.), CA42978, CA52072, and CA63139 (to C. J. D.); by funds from the Indiana University School of Medicine Diabetes Research and Training Center and the Grace M. Showalter trust (to L. A. Q.); and by the National Cancer Institute, under contract with ABL. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PTK

protein-tyrosine kinase

GST

glutathione S-transferase

PKN

protein kinase N(ovel)

PRK

protein kinase C-related kinase

SH

Src homology domain

WASP

Wiskott-Aldrich syndrome protein

RFLP

restriction fragment length polymorphism

kb

kilobase pair(s)

bp

base pair(s)

SRF

serum response factor.
↵²L. A. Quilliam, L. A. Mickelson-Young, and J. K. Westwick, unpublished observations.
↵³Bokoch, G. M., Wang, Y., Bohl, B. P., Sells, M. A., Quilliam, L. A., and Knaus, U. G. (1996) J. Biol. Chem. 271, 25746-25749.
- Received June 20, 1996.
- Revision received September 16, 1996.