Direct activation of the fission yeast PAK Shk1 by the novel SH3 domain protein, Skb5.
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.
Yang
P
P
Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Pimental
R
R
Lai
H
H
Marcus
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D054462:p21-Activated Kinases; D018909:src Homology Domains / Q000235:genetics
D002352:Carrier Proteins; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D029702:Schizosaccharomyces pombe Proteins; C403123:Skb5 protein, S pombe; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; C094341:shk1 protein, S pombe