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J Biol Chem, Vol. 274, Issue 51, 36052-36057, December 17, 1999

Direct Activation of the Fission Yeast PAK Shk1 by the Novel SH3 Domain Protein, Skb5^*

Peirong Yang, Ruth Pimental, Hong Lai, and Stevan Marcus^§

From the Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

The p21-activated kinase (PAK) homolog Shk1 is essential for cell viability in the fission yeast Schizosaccharomyces pombe. Roles have been established for Shk1 in the regulation of cell morphology, sexual differentiation, and mitosis in S. pombe. In this report, we describe the genetic and molecular characterization of a novel SH3 domain protein, Skb5, identified as a result of a two-hybrid screen for Shk1 interacting proteins. S. pombe cells carrying a deletion of the skb5 gene exhibit no discernible phenotypic defects under normal growth conditions, but when subjected to hypertonic stress, become spheroidal in shape and growth impaired. Both of these defects can be suppressed by overexpression of the Shk1 modulator, Skb1. The growth inhibition that results from overexpression of Shk1 in S. pombe cells is markedly suppressed by a null mutation in the skb5 gene, suggesting that Skb5 contributes positively to the function of Shk1 in vivo. Consistent with this notion, we show that Skb5 stimulates Shk1 catalytic function in S. pombe cells. Furthermore, and perhaps most significantly, we show that bacterially expressed recombinant Skb5 protein directly stimulates the catalytic activity of recombinant Shk1 kinase in vitro. These and additional data described herein demonstrate that Skb5 is a direct activator of Shk1 in fission yeast.

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^* This work was supported by National Institutes of Health Grant R01GM53239. DNA sequencing was performed by the University of Texas M. D. Anderson Cancer Center Core DNA Sequencing Facility, which is supported by National Institutes of Health Grant P30CA16672.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF192549 (for skb5).

These authors contributed equally to this work.

^§ To whom correspondence should be addressed. Tel.: 713-745-2032; Fax: 713-794-4394; E-mail: smarcus@notes.mdacc.tmc.edu.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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