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.

Regulated interactions between microtubules (MTs) and the cell cortex control MT dynamics and position the mitotic spindle. In eukaryotic cells, the adenomatous polyposis coli/Kar9p and dynein/dynactin pathways are involved in guiding MT plus ends and MT sliding along the cortex, respectively. Here we identify Bud14p as a novel cortical activator of the dynein/dynactin complex in budding yeast. Bud14p accumulates at sites of polarized growth and the mother-bud neck during cytokinesis. The localization to bud and shmoo tips requires an intact actin cytoskeleton and the kelch-domain-containing proteins Kel1p and Kel2p. While cells lacking Bud14p function fail to stabilize the pre-anaphase spindle at the mother-bud neck, overexpression of Bud14p is toxic and leads to elongated astral MTs and increased dynein-dependent sliding along the cell cortex. Bud14p physically interacts with the type-I phosphatase Glc7p, and localizes Glc7p to the bud cortex. Importantly, the formation of Bud14p–Glc7p complexes is necessary to regulate MT dynamics at the cortex. Taken together, our results suggest that Bud14p functions as a regulatory subunit of the Glc7p type-I phosphatase to stabilize MT interactions specifically at sites of polarized growth

Here, we report the identi®cation and characterization of a new member of the RalGDS-family, which is widely expressed and interacts strongly and selectively with the GTP-bound forms of M-Ras and p21 Ras. This Ras pathway modulator (RPM), also termed RGL3, exhibited Ras-binding and catalytic domains typical of the RalGDS-family of guanine nucleotide exchange factors, and was most similar to Rlf (RalGDS-like factor), but was distinguished by a unique proline-rich region with multiple candidate SH3-domain binding sites. RPM/ RGL3 resembled AF-6 and Nore1 in interacting strongly with constitutively active M-Ras and p21 Ras. In contrast to Rlf, transiently expressed RPM/RGL3 did not activate an Elk-1-inducible reporter gene alone or in combination with activated p21 Ras, but strongly inhibited induction of this reporter gene by co-expression of activated H-Ras or MEKK-1. This inhibitory eect was independent of the Ras binding domain and required a second signal provided by p21 Ras or MEKK-1, but not Raf-1 or M-Ras. Expression of RPM/RGL3 also strongly inhibited cell growth of ®broblasts transformed by an activated Src Y527F. Thus, RPM/RGL3 is a novel potential eector of both p21 Ras and M-Ras with the novel function of negatively regulating Elk-1-dependent gene induction downstream of p21 Ras or MEKK-1. Oncogene (2001) 20, 188 ± 197.

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