In this study we have readdressed the signal transduction role of the Cdc42-binding (CRIB) domain in Ste20, a yeast PAK family kinase. We provide genetic and biochemical evidence that Cdc42-Ste20 binding regulates Ste20 kinase activity and signaling competence. Point mutations in the CRIB domain decrease pheromone response to a degree proportional to their Cdc42-binding defect (Fig. ), suggesting that the Cdc42-Ste20 interaction is normally critical for pheromone signaling. This conclusion contrasts with that made previously from studies in which larger deletions were used to remove the Cdc42-binding site (27
). In retrospect, those studies appear to have bypassed the Cdc42 requirement (39
) rather than show it is unnecessary, because the deletions also removed inhibitory residues immediately downstream of the CRIB consensus motif.
Indeed, we found here that the Ste20 CRIB domain interacts with the kinase domain and disruption of this interaction by mutation relieves the requirement for Cdc42 binding (Fig. ). Our results overwhelmingly support a model in which Cdc42 activates Ste20 by antagonizing the negative influence of sequences within the CRIB domain (see Fig. and ). They also complement the recent data for Cdc42 mutants that show defects in both Ste20 binding and pheromone response (39
) and are consistent with other recent studies on PAKs from other organisms (9
). We suggest that the basic mechanism proposed for GTPase-mediated regulation of PAKs (reviewed in reference 21
) is shared by Ste20, indicating that Ste20 continues to serve as a model for PAK function. Our results with Ste20 are equally compatible with models in which autoinhibition occurs intramolecularly (in cis
) or intermolecularly between members of a homodimer (in trans
), as shown recently for mammalian PAK1 (44
FIG. 7. General model for the production and utilization of activated Ste20. Based on results in this study, the model proposes that GTP-bound Cdc42 (presumably activated by its exchange factor, Cdc24) converts Ste20 to an active form (asterisk) by antagonizing (more ...)
Interestingly, PAKs are not alone among targets of Rho family GTPases in being regulated by conversion between autoinhibited and uninhibited forms, generically termed intrasteric regulation (25
). Instead, this mechanism appears to be common among many kinase and nonkinase targets of Rho, Rac, and Cdc42 (7
). It is also notable that the yeast pheromone response pathway (for a review, see reference 15
) repeatedly uses a strategy in which positive activation is accomplished by antagonism of a negative regulator: (i) ligand-bound receptor activates Gβγ by inhibiting the negative effect of the Gα subunit; (ii) Cdc42 activates Ste20 by inhibiting the negative effect of the Ste20 CRIB domain; (iii) Ste20 activates Ste11 by inhibiting the negative effect of the Ste11 N terminus; (iv) Fus3 and Kss1 activate Ste12-mediated transcription by inhibiting repressors of Ste12, Dig1/Rst1, and Dig2/Rst2; and (v) release of an interaction between the N and C termini of Ste5 may also contribute to activation of the MAP kinase cascade (54
We observed hyperactive kinase activity in all Ste20 mutants with Cdc42-independent signaling ability (Fig. ). The level of hyperactivity (three- to eightfold) is similar to that observed with another yeast PAK, Cla4 (6
), whereas each of these is relatively modest compared to mammalian PAKs bearing analogous mutations (9
). While our results are consistent with those for other PAKs, they were not observed in previous studies of Ste20 mutants lacking the CRIB domain (27
). It is not clear why our results were different, though they were consistent regardless of whether an N-terminal GFP tag or a C-terminal Myc tag was used to purify Ste20. It is possible that in prior studies, the wild-type protein was artificially activated by unfolding to the “open” conformation, either during preparation of cell lysates or by binding of polyclonal anti-Ste20 antibodies (27
); alternatively, overexpression may have made Ste20 resistant to negative regulation (45
). It is also conceivable that in our experiments the wild-type protein became inactivated during lysate preparation by a mechanism to which the Δ334-369 mutant is insensitive (e.g., refolding); if so, this may have simultaneously obscured differences between the wild type and the signaling-deficient mutants (e.g., S338A/H345G).
Regardless of the explanation, our results indicate that there are measurable differences in kinase properties between wild-type Ste20, signaling-deficient mutants, and Cdc42-independent mutants which are likely to be of fundamental importance to signaling. The toxicity resulting from overexpression of the hyperactive mutants (Fig. ) independently suggests that the kinase hyperactivity observed in vitro reflects real changes in kinase properties in vivo.
We also found that Ste20 kinase activity is stimulated by expression of GTP-bound Cdc42 in vivo. This has not been reported previously for S. cerevisiae
; stimulation in vitro was observed using baculovirus-produced proteins (58
), but this was not reproduced using Ste20 purified from yeast extracts (27
). Most importantly, we found that the signaling defect of the S338A/H345G mutant correlated with an inability to respond to Cdc42 stimulation, while the ability of L369G or Δ334-369 mutations to confer Cdc42-independent signaling correlated with deregulated, Cdc42-independent kinase activity. Thus, our observations link the Cdc42 dependence of Ste20 kinase activity to the in vivo signaling behavior and argue that signaling by wild-type Ste20 requires that its kinase activity be stimulated by Cdc42.
How is Ste20 kinase activity harnessed by the mating pathway? It is informative that, despite their hyperactive kinase activity, the Cdc42-independent alleles Δ334-369, L369G, and S338A/H345G/L369G do not show constitutive signaling—i.e., there is no increase in either the basal or induced levels of FUS1-lacZ
(Fig. and data not shown; see also reference 27
). While this may appear counterintuitive, it is in fact consistent with our previous arguments (39
) that the rate-limiting step in pheromone signaling is unlikely to be the production of active Ste20, but rather the access of Ste20 to its substrate—namely, the MAP kinase kinase kinase Ste11 associated with the scaffold protein Ste5 (18
)—perhaps along with other events, such as conformational changes in Ste5 (54
). Consistent with this view, there is no indication that pheromone can stimulate Ste20 kinase activity, as noted previously (65
) and confirmed here in parallel with experiments in which Ste20 kinase activity clearly could be stimulated by another method, expression of Cdc42Q61L
(Fig. ). The fact that pheromone did not mimic Cdc42Q61L
expression may also indicate that the levels of GTP-bound Cdc42 are not increased by pheromone, despite the known ability of Gβγ to assemble with the Cdc42 exchange factor, Cdc24. While association of Gβγ with Cdc24 helps guide cell polarization along pheromone gradients (11
), it is unresolved whether Gβγ alters only the localization or also the activity levels of Cdc24 and Cdc42, but currently there is no evidence for the latter.
It remains conceivable that pheromone effects on Cdc24, Cdc42, and Ste20 activities do occur but are difficult to detect because they either are extremely labile (e.g., GTP hydrolysis) or involve only a small fraction of molecules. Nevertheless, it seems clear that assembly of complexes involving Gβγ and Cdc24 is not necessary for pheromone to induce Ste20-dependent signaling, as mutants in which these complexes are disrupted are fully competent at pheromone response (41
). Furthermore, we show here (Fig. and ) that the effects of disrupting the Cdc42-Ste20 interaction are apparent without pheromone stimulation and in the absence of Gβ (Ste4), which, along with other observations (39
), suggests that the Cdc42-dependent step normally precedes pathway activation rather than requiring regulation by pheromone or Gβγ. Therefore, in total these findings indicate that although both the Cdc42-Ste20 interaction and Ste20 kinase activity are required for pheromone-dependent signaling, pheromone stimulation of these states is neither required nor evident.
The simplest model would seem to be that pheromone regulates the ability of an existing pool of active Ste20 to activate the downstream MAP kinase cascade (Fig. ). It is noteworthy in this regard that overexpression of Ste20ΔN, an “activated” form of Ste20 lacking its N-terminal 495 residues, induces FUS1-lacZ
to relatively miniscule levels compared to the level when pheromone is added (27
); this suggests that pheromone still triggers a critical rate-limiting event, such as phosphorylation of Ste11 by Ste20 (60
) or expedited signal transmission from activated Ste11 (18
Related to the issue of how pathway stimuli harness Ste20 activity, our observations suggest that the role of the Cdc42-Ste20 interaction is not qualitatively distinct between the mating and filamentation pathways, in contrast to previous conclusions (27
). Instead, we found that the two pathways are impacted similarly by CRIB domain mutations: (i) precise disruption of the Cdc42-Ste20 interaction by point mutation causes a severe reduction in function, and (ii) these defects are suppressed by either the L369G point mutation or the complete CRIB domain deletion, though not to wild-type efficacy for either pathway. These observations indicate that both pathways require Cdc42 to bind Ste20 and that in each pathway a primary role for this binding is to antagonize the negative effect of the Ste20 CRIB domain (Fig. ). In addition, when the CRIB domain is completely removed from Ste20, producing a hyperactive kinase, signaling in both pathways is not hyperactive but instead is demonstrably less efficient (to a degree that is exaggerated by reduced expression). It seems likely that this handicap is due to delocalization from the plasma membrane, where activation of the mating pathway MAP kinase cascade is thought to be initiated (36
). Whether activation of the filamentation pathway MAP kinase cascade is also initiated at the membrane is less clear, though interactions of Ste11 and Ste7 kinases with the polarity proteins Spa2 and Sph1 (52
) could potentially play a role in restricting filamentation pathway signaling to the cell periphery. Proper localization is also critical to the essential function shared by Ste20 and Cla4, as removal of the Cdc42-binding site disrupts this function in either kinase (6
) (Fig. ). Relatedly, excess levels of hyperactive Ste20 are lethal (27
) (Fig. ) and produce a depolarized actin phenotype (27
), similar to when Ste20 and Cla4 are absent (22
) or when Cdc42 is inactivated (1
). This requirement for not only PAK activity per se but also its proper spatial regulation is consistent with the fundamental asymmetry of cytoskeletal reorganization events regulated by Cdc42 during cell growth and division (23
are detectably impaired for signaling (e.g., in comparison to the Ste20L369G
form, which retains Cdc42-binding; cf. Fig. versus and see Fig. ), it is somewhat surprising how well they do signal, given their delocalization. It is conceivable that the kinase hyperactivity of these forms compensates for their delocalization to an extent that is purely coincidental. It is also possible that these forms are still restricted to signaling in specialized subcellular locales, in a manner assisted by interactions with other proteins. Indeed, the signaling efficiency of Ste20Δ334-369
appears to depend on interaction with the SH3 domain protein Bem1, as it is reduced by deletion of BEM1
) and by mutation of a Bem1-binding domain within Ste20 (M. J. Winters and P. M. Pryciak, unpublished data). This may indicate that while Ste20Δ334-369
is delocalized, it might still signal predominantly at the cell periphery, where Bem1 is abundant (3
). Ste20 also binds the pheromone-activated Gβγ complex (29
), potentially allowing recruitment of Ste20 to its mating pathway substrates in a manner that compensates to some degree for defects in localization via Cdc42.
Further work will be required to determine the full spectrum of mechanisms involved in colocalizing activated Ste20 with its substrates and to describe the complete set of events triggered by a specific pathway stimulus that are rate limiting for Ste20-dependent signal transduction.