Phosphorylation of Bem2p and Bem3p may contribute to local activation of Cdc42p at bud emergence

Abstract

Site-specific activation of the Rho-type GTPase Cdc42p is critical for the establishment of cell polarity. Here we investigated the role and regulation of the GTPase-activating enzymes (GAPs) Bem2p and Bem3p for Cdc42p activation and actin polarization at bud emergence in Saccharomyces cerevisiae. Bem2p and Bem3p are localized throughout the cytoplasm and the cell cortex in unbudded G1 cells, but accumulate at sites of polarization after bud emergence. Inactivation of Bem2p results in hyperactivation of Cdc42p and polarization toward multiple sites. Bem2p and Bem3p are hyperphosphorylated at bud emergence most likely by the Cdc28p-Cln2p kinase. This phosphorylation appears to inhibit their GAP activity in vivo, as non-phosphorylatable Bem3p mutants are hyperactive and interfere with Cdc42p activation. Taken together, our results indicate that Bem2p and Bem3p may function as global inhibitors of Cdc42p activation during G1, and their inactivation by the Cdc28p/Cln kinase contributes to sitespecific activation of Cdc42p at bud emergence.

Yeast and fungal morphogenesis from an evolutionary perspective

“…A non-phosphorylatable mutant of Rga2 acts in a dominant-active fashion and inhibits cell polarization upon overexpression [95]. The same holds true for the respective Bem3 mutants even for low levels of expression [90]. These results provide compelling evidence that Cdk1 activation at G1-S induces polarization at least in part through inhibition of Cdc42 GAP.…”

“…Two proteomics searches for proteins associated with specific Cdc28-cyclin complexes identified Rga1 and Bem3 as targets of Cdc28 in complex with G1 cyclin Gln2 [92,93]. Three recent reports involving S. cerevisae and one involving C. albicans provided evidence that the GAPs are phosphorylated by Cdc28 and Pho85 Cdks in late G1 [90,[94][95][96]. Interestingly, overexpression of a GAP-deficient allele of Bem3 acts in a dominant-negative fashion and induces spontaneous polarization of G1 cells in a manner similar to constitutively active Cdc42 [90].…”

“…Three recent reports involving S. cerevisae and one involving C. albicans provided evidence that the GAPs are phosphorylated by Cdc28 and Pho85 Cdks in late G1 [90,[94][95][96]. Interestingly, overexpression of a GAP-deficient allele of Bem3 acts in a dominant-negative fashion and induces spontaneous polarization of G1 cells in a manner similar to constitutively active Cdc42 [90]. This suggests that Cdc42's intrinsic nucleotide exchange is sufficient to achieve a threshold level of Cdc42 GTP once the GAPs are inactivated.…”

“…Another GAP, Bem2, shows GAP activity towards Rho1 instead of Cdc42 in vitro [89]. However, genetic analysis of cell polarity indicates that Bem2 might still act on Cdc42 in living cells without relying on its GAP activity [90,91]. Genetic studies showed that the Cdc42 GAPs have overlapping but non-identical functions.…”

Fungal Morphogenesis

“…Therefore, hypha-specific expression of Hgc1 is responsible for maintaining Cdc42-GTP at the hyphal tips and for sustained polarized growth in hyphae. In S. cerevisiae, Bem3 and Rga2 GAPs are hypophosphorylated at the time of bud emergence by Cdk1 G1 , and the inhibition of GAP activity by Cdk1 G1 phosphorylation leads to increased amounts of Cdc42-GTP and actin polarization for bud emergence (Knaus et al 2007;Sopko et al 2007). Therefore, inhibition of GAPs via phosphorylation by G 1/ CDKs may be a conserved mechanism that promotes polarized growth in fungi (Wang 2009).…”