Shk1, a homolog of the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases, is a component of a Ras/Cdc42 signaling module in the fission yeast Schizosaccharomyces pombe.

Abstract

We describe a protein kinase, Shkl, from the fission yeast Schizosaccharomyces pombe, which is structurally related to the Saccharomyces cerevisiae Ste2O and mammalian p65PAK protein kinases. We provide genetic evidence for physical and functional interaction between Shkl and the Cdc42 GTP-binding protein required for normal cell morphology and mating in S. pombe. We further show that expression of the STE20 gene complements the shki null mutation and that Shkl is capable of signaling to the pheromone-responsive mitogen-activated protein kinase cascade in S. cerevisiae. Our results lead us to propose that signaling modules composed of small GTP-binding proteins and protein kinases related to Shkl, Ste2O, and p65PAK, are highly conserved in evolution and participate in both cytoskeletal functions and mitogen-activated protein kinase signaling pathways.ras genes are highly conserved in evolution and encode small GTP-binding proteins that regulate cell growth and differentiation in a broad spectrum of eukaryotic organisms (1). The fission yeast Schizosaccharomyces pombe possesses a single known ras homolog, rasl, the product of which is required for at least two distinct cellular functions. First, Rasl is required for sexual differentiation-namely, conjugation and sporulation-which is induced by starvation and by peptide mating pheromones that bind to serpentine receptors (2-4). Rasl functions upstream of a mitogen-activated protein (MAP) kinase (MAPK) In this report, we describe a protein kinase, Shk,1, and provide evidence that it mediates functions of the Rasl/Cdc42 signaling complex in S. pombe. Shkl is highly related in structure to the Ste2O kinase, which is required for sexual response in Saccharomyces cerevisiae (19,20), and to the mammalian Cdc42/Racl-binding kinase, p65PAK (Pak) (21).Our results lead us to propose that signaling pathways mediated by small GTP-binding proteins and protein kinases related to Shkl are conserved in evolution and participate in regulation of the cytoskeleton and MAPK modules.MATERIALS AND METHODS Microbial Manipulation and Analysis. S. pombe strains SP870 (h9O ade6-210 leul-32 ura4-D18) and SP66 (h9o ade6-216 leul-32) were provided by D. Beach (Cold Spring Harbor Laboratory). SP870D (h90 ade6-210/ade6-210 leul-32/leul-32 ura4-D18/ura4-D18) is a spontaneous diploid derived from SP870 (V. Jung, personal communication). SP206U (h90 ade6-210/ade6-210 leul-32/leul-32 ura4-D18/ura4-D18 shk1::ura4/shk1) was constructed by transformation of SP870D with an Ec1136II-Msc I fragment of shkl::ura4 from plasmid pBSSHK1::URA4. SP206UA (h90 ade6-210/ade6-210 leul-32/leul-32 ura4-D18/ ura4-D18 shk1::ura4:.ADE2/shk1+) was constructed by transforming SP206U with a Not I fragment of ura4:.ADE2 obtained from pVIN (22). SP42N17 (h90 ade6-210 leul-32 ura4::adh1-cdc42[T17N]-ADE2) was constructed by transforming the S.Abbreviations: MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; GST, glutathione Stransferase.

A novel human SPS1/STE20 homologue, KHS, activates Jun N-terminal kinase

“…The speci®city of the antiserum was veri®ed, since immunoprecipitation was blocked when the antiserum was preincubated with recombinant GST-KHS ( Figure 3a). This antiserum was also able to speci®cally immunoprecipitate a 95 kD protein from metaboli- (Katz et al, 1994;Creasy and Cherno , 1995;Cvrckova et al, 1995;Leberer et al, 1992;Ramer and Davis, 1993;Freisen et al, 1994;Marcus et al, 1995;Bagrodia et al, 1995;Knaus et al, 1995;Manser et al, 1994Martin et al, 1995;Ottilie et al, 1995). Catalytic domains are boxed.…”

Signaling mechanisms that regulate actin‐based motility processes in the nervous system

“…Phosphorylated PAK is then released, presumably to activate downstream signaling cascades (Manser et al 1994). PAK is involved in a number of processes, including MAP kinase signaling (Marcus et al 1995), leukocyte activation (Knaus et al 1995), and JNK/SAPK activation (Bagrodia et al 1995;Frost et al 1996). A role for PAK in cytoskeletal processes is evident, as PAK localizes to membrane ruffles in response to growth factors and activated rac (Dharmawardhane et al 1997).…”

YSK1, a novel mammalian protein kinase structurally related to Ste20 and SPS1, but is not involved in the known MAPK pathways

“…Cla4 interacts with Cdc42, a small G-protein (or GTPase) localized at the bud site (Cvrckova et al, 1995). In ®ssion yeast, Shk1 has been identi®ed as a Ste20 homologue (Marcus et al, 1995). Shk1 forms a complex with Cdc42, and a functional interaction between Shk1, Cdc42, and Ras1 is required for normal cell morphology and mating.…”