Pheromone Signalling in Saccharomyces cerevisiae Requires the Small GTP-Binding Protein Cdc42p and Its Activator CDC24

Abstract

Pheromone signalling in Saccharomyces cerevisiae is mediated by the STE4-STE18 G-protein ␤␥ subunits. A possible target for the subunits is Ste20p, whose structural homolog, the serine/threonine kinase PAK, is activated by GTP-binding p21s Cdc42 and Rac1. The putative Cdc42p-binding domain of Ste20p, expressed as a fusion protein, binds human and yeast GTP-binding Cdc42p. Cdc42p is required for ␣-factor-induced activation of FUS1. cdc24 ts strains defective for Cdc42p GDP/GTP exchange show no pheromone induction at restrictive temperatures but are partially rescued by overexpression of Cdc42p, which is potentiated by Cdc42p 12V mutants. Epistatic analysis indicates that CDC24 and CDC42 lie between STE4 and STE20 in the pathway. The two-hybrid system revealed that Ste4p interacts with Cdc24p. We propose that Cdc42p plays a pivotal role both in polarization of the cytoskeleton and in pheromone signalling.In eukaryotic cells, many growth and differentiation signals are channelled through protein phosphorylation cascades that involve the mitogen-activated protein (MAP) kinase family. The similarity of MAP kinase cascades in multicellular organisms to those in the yeast Saccharomyces cerevisiae is a subject of great interest (7,17,42,50). One of the best studied of these cascades at the genetic level is the pheromone-dependent pathway in S. cerevisiae, which links occupation of pheromone receptors by a-or ␣-factor to a variety of events that lead to mating (for reviews, see references 3, 27, and 41). The pheromone-induced transcriptional activation of mating-specific genes requires a kinase cascade that includes the protein kinases encoded by STE20, STE11, STE7, KSS1, and FUS3. Activation of the pathway is initiated by the heterotrimeric G-protein ␤␥ subunits encoded by STE4 and STE18 (71, 72); the STE20 gene product is a kinase which may lie at the top of the kinase cascade and has been implicated as a target of the STE4-encoded ␤ subunit (32). In mammalian cells, the activation of MAP kinase by various proto-oncogenic tyrosine kinases requires small GTP-binding proteins (p21s) of the Ras family; more recently, heterotrimeric G-protein signalling to MAP kinase has been shown to be mediated by ␤␥ subunits via Ras (13). While the mating pathway in Schizosaccharomyces pombe parallels this pathway in requiring ras1 (73), there has been no evidence for the involvement of a p21 in the S. cerevisiae pheromone-induced kinase cascade.We have described a number of proteins that appear to be the target of the Rho subfamily of p21s (39,40). One of these proteins was purified and shown to be a serine/threonine kinase that is directly activated by GTP-binding Cdc42 (GTPCdc42p) and GTP-Rac1. This brain-enriched kinase, designated PAK (40), is related to Ste20p both in the kinase domain and in a separate region identified as the p21-binding domain. These findings suggested that Cdc42p, a p21 first identified as a gene product required for polarized budding (29), participates in the mating response (Rac has not been found in S. cere...

Characterization of Brx, a novel Dbl family member that modulates estrogen receptor action

“…Inhibition of estrogen receptor activation by N17Cdc42Hs, coupled with the presence of a DH (GEF) region and a receptor-binding region, suggests that Brx may function as a molecular bridge between transmembrane signaling pathways and nuclear receptor signaling (see below). In keeping with this postulate, Cdc42Hs, has been implicated in seven transmembrane growth factor receptor signaling in both NIH3T3 cells (Nobes and Hall, 1995) and yeast (Zhao et al, 1995). We note that binding of Brx to ER may not precisely correlate with the ability of Brx to augment estrogendependent reporter activity, since transfection of a Brx expression mutant similar to the DC1 fragment that failed to bind ER (Figure 4d) did cause a slight increase in estrogen-dependent reporter activity (not shown).…”

Induction of apoptosis by SLK, a Ste20-related kinase

“…Database searches revealed that the cloned cDNA encodes a protein kinase highly related to LOK, a Ste20-related protein kinase preferentially expressed in lymphocytes (Kuramochi et al, 1997). Analysis of the kinase domain shows that SLK is also related to MST1/2 (Creasy and Cherno , 1995; Katoh et al, 1995;Schinkmann and Blenis, 1997) and more distantly related to Ste20, a yeast kinase involved in the pheromone response pathway (Zhao et al, 1995). Furthermore, SLK showed 70% identity to M-NAP and 63 and 71% identity to AT1-46 in two distinct carboxy terminal domains.…”

“…In yeast, the Ste20 serine/threonine protein kinase regulates a mitogen-activated protein kinase pathway consisting in Ste11 (MEK kinase), Ste7 (MEK) and Fus3/Kss1 (MAPK) protein kinases involved in the control of mating response (Zhao et al, 1995). Alpha factor binding to its G-protein coupled receptor results in the downstream activation of Ste20 by the bg complex released from the heterotrimeric G protein.…”

Cell growth control by G protein-coupled receptors: from signal transduction to signal integration

“…Free bg dimers then activate a serine-threonine kinase, Ste20, stimulating the activity of a linear cascade of kinases including sequentially Ste11 and Ste7, which phosphorylate and activate the yeast MAPK homolog Fus3 and Kss1. Extensive search for molecules linking yeast bg complexes to Ste20 has led to the recent discovery that Cdc42 participates in Ste20 activation (Simon et al, 1995) and that Ste4 might directly bind and activate Cdc24, a nucleotide exchange factor for Cdc42 (Zhao et al, 1995). Thus, in yeast, the G protein b subunit can initiate activity of a MAPK cascade by binding an exchange factor for the Cdc42 GTPase, and then this GTP-binding protein physically interacts with the most upstream kinase, Ste20, causing its activation (Herskowitz, 1995).…”