The highly conserved skb1 gene encodes a protein that interacts with Shk1, a fission yeast Ste20/PAK homolog

The highly conserved skb1 gene encodes a protein that interacts with Shk1, a fission yeast Ste20/PAK homolog

^*Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030; ^‡Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235; and ^§Department of Protein Structure, Amgen Inc., Thousand Oaks, CA 91320-1789

Abstract

The Shk1 protein kinase, a homolog of Saccharomyces cerevisiae Ste20 and mammalian p21^Cdc42/Rac-activated kinases, is an essential component of a Ras- and Cdc42-dependent signaling cascade required for cell viability, normal morphology, and mitogen-activated protein kinase-mediated sexual responses in the fission yeast, Schizosaccharomyces pombe. To identify S. pombe proteins that modulate or mediate Shk1 functions, we conducted a two-hybrid screen for Shk1-interacting proteins. One of the genes identified as a result of this screen was skb1. We show that Skb1 interacts with a region of the N-terminal regulatory domain of Shk1 distinct from that to which Cdc42 binds, and that Shk1, Cdc42, and Skb1 are able to form a ternary complex in vivo. S. pombe cells carrying an skb1 null mutation are less elongate in morphology than wild-type cells and exhibit a moderate growth defect. The morphology defect of the skb1 deletion mutant is suppressed by overexpression of Shk1. Overexpression of Skb1 causes wild-type S. pombe cells to become hyperelongated. Additional genetic analyses described herein suggest that Skb1 is a component of the morphology control branch of the Ras signaling cascade in S. pombe and that it positively modulates Shk1 function. Homologs of Skb1 are encoded by open reading frames in the genomes of S. cerevisiae and Caenorhabditis elegans and by an uncharacterized human cDNA sequence. Thus, skb1 may be the first well-characterized member of a highly conserved family of genes encoding potential p21^Cdc42/Rac-activated kinase regulators.

Footnotes

↵ † M.G. and P.Y. contributed equally to this work.
↵ ¶ To whom reprint requests should be addressed. e-mail: stevan_marcus{at}molgen.mda.uth.tmc.edu.
Michael H. Wigler, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
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Abbreviations: GAD, Gal4 transcriptional activation domain; GBD, Gal4 DNA binding domain; LBD, LexA DNA-binding domain; MAPK, mitogen-activated protein kinase; MAPKKK, MAPK kinase kinase; β-gal, β-galactosidase; PAK, p21^Cdc42/Rac-activated kinase.

Data deposition: The sequence reported in this paper has been deposited in the GenBank data base (accession no. U59684U59684).