Myosin I is required for hypha formation in Candida albicans.

The pathogenic yeast Candida albicans can undergo a dramatic change in morphology from round yeast cells to long filamentous cells called hyphae. We have cloned the CaMYO5 gene encoding the only myosin I in C. albicans. A strain with a deletion of both copies of CaMYO5 is viable but cannot form hyphae under all hypha-inducing conditions tested. This mutant exhibits a higher frequency of random budding and a depolarized distribution of cortical actin patches relative to the wild-type strain. We found that polar budding, polarized localization of cortical actin patches, and hypha formation are dependent on a specific phosphorylation site on myosin I, called the "TEDS-rule" site. Mutation of this serine 366 to alanine gives rise to the null mutant phenotype, while a S366D mutation, the product of which mimics a phosphorylated serine, allows hypha formation. However, the S366D mutation still causes a depolarized distribution of cortical actin patches in budding cells, similar to that in the null mutant. The localization of CaMyo5-GFP together with cortical actin patches at the bud and hyphal tips is also dependent on serine 366. Intriguingly, the cortical actin patches in the majority of the hyphae of the mutant expressing Camyo5(S366D) were depolarized, suggesting that although their distribution is dependent on myosin I localization, polarized cortical actin patches may not be required for hypha formation.

Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans.

The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. We have cloned the CaCDC35 gene encoding C. albicans adenylyl cyclase by functional complementation of the conditional growth defect of Saccharomyces cerevisiae cells with mutations in Ras1p and Ras2p. It has previously been shown that these Ras homologues regulate adenylyl cyclase in yeast. The C. albicans adenylyl cyclase is highly homologous to other fungal adenylyl cyclases but has less sequence similarity with the mammalian enzymes. C. albicans cells deleted for both alleles of CaCDC35 had no detectable cAMP levels, suggesting that this gene encodes the only adenylyl cyclase in C. albicans. The homozygous mutant cells were viable but grew more slowly than wild-type cells and were unable to switch from the yeast to the hyphal form under all environmental conditions that we analyzed in vitro. Moreover, this morphogenetic switch was completely blocked in mutant cells undergoing phagocytosis by macrophages. However, morphogenetic switching was restored by exogenous cAMP. On the basis of epistasis experiments, we propose that CaCdc35p acts downstream of the Ras homologue CaRas1p. These epistasis experiments also suggest that the putative transcription factor Efg1p and components of the hyphal-inducing MAP kinase pathway depend on the function of CaCdc35p in their ability to induce morphogenetic switching. Homozygous cacdc35 Delta cells were unable to establish vaginal infection in a mucosal membrane mouse model and were avirulent in a mouse model for systemic infections. These findings suggest that fungal adenylyl cyclases and other regulators of the cAMP signaling pathway may be useful targets for antifungal drugs.

Ras links cellular morphogenesis to virulence by regulation of the MAP kinase and cAMP signalling pathways in the pathogenic fungus Candida albicans.

The pathogenic fungus Candida albicans is capable of responding to a wide variety of environmental cues with a morphological transition from a budding yeast to a polarized filamentous form. We demonstrate that the Ras homologue of C. albicans, CaRas1p, is required for this morphological transition and thereby contributes to the development of pathogenicity. However, CaRas1p is not required for cellular viability. Deletion of both alleles of the CaRAS1 gene caused in vitro defects in morphological transition that were reversed by either supplementing the growth media with cAMP or overexpressing components of the filament-inducing mitogen-activated protein (MAP) kinase cascade. The induction of filament-specific secreted aspartyl proteinases encoded by the SAP4-6 genes was blocked in the mutant cells. The defects in filament formation were also observed in situ after phagocytosis of C. albicans cells in a macrophage cell culture assay and, in vivo, after infection of kidneys in a mouse model for systemic candidiasis. In the macrophage assay, the mutant cells were less resistant to phagocytosis. Moreover, the defects in filament formation were associated with reduced virulence in the mouse model. These results indicate that, in response to environmental cues, CaRas1p is required for the regulation of both a MAP kinase signalling pathway and a cAMP signalling pathway. CaRas1p-dependent activation of these pathways contributes to the pathogenicity of C. albicans cells through the induction of polarized morphogenesis. These findings elucidate a new medically relevant role for Ras in cellular morphogenesis and virulence in an important human infectious disease.

Molecular interactions of the Gbeta binding domain of the Ste20p/PAK family of protein kinases. An isolated but fully functional Gbeta binding domain from Ste20p is only partially folded as shown by heteronuclear NMR spectroscopy.

The transmission of the mating signal of the budding yeast Saccharomyces cerevisiae requires Ste20p, a member of the serine/threonine protein kinases of the Ste20p/PAK family, to link the Gbeta subunit of the heterotrimeric G protein to the mitogen-activated protein kinase cascades. The binding site of Ste20p to the Gbeta subunit was mapped to a consensus sequence of SSLphiPLI/VXphiphibeta (X for any residue; phi for A, I, L, S or T; beta for basic residues), which was shown to be a novel Gbeta binding (GBB) motif present only in the noncatalytic C-terminal domains of the Ste20p/PAK family of protein kinases (Leeuw, T., Wu, C., Schrag, J. D., Whiteway, M., Thomas, D. Y., and Leberer, E. (1998) Nature 391, 191-195; Leberer, E., Dignard, D., Thomas, D. Y., and Leeuw, T. (2000) Biol. Chem. 381, 427-431). Here, we report the results of an NMR study on two GBB motif peptides and the entire C-terminal domain derived from Ste20p. The NMR data show that the two peptide fragments are not uniquely structured in aqueous solution, but in the presence of 40% trifluoroethanol, the longer 37-residue peptide exhibited two well defined, but flexibly linked helical structure elements. Heteronuclear NMR data indicate that the fully functional 86-residue C-terminal domain of Ste20p is again unfolded in aqueous solution but has helical secondary structure preferences similar to those of the two peptide fragments. The NMR results on the two GBB peptides and the entire GBB domain all indicate that the two important binding residues, Ser(879) and Ser(880), are located at the junction between two helical segments. These experimental observations with the prototype GBB domain of a novel family of Gbeta-controlled effectors may have important implications in understanding the molecular mechanisms of the signal transduction from the heterotrimeric G protein to the mitogen-activated protein kinase cascade.

A comprehensive analysis of gene expression profiles in a yeast N-glycosylation mutant.

Although protein N-glycosylation is critical to many cell functions, its downstream targets remain largely unknown. In all eukaryotes, N-glycosylation utilizes the lipid-linked oligosaccharide (LLO) precursor, whose synthesis is initiated by the ALG7 gene. To elucidate the key signaling and metabolic events affected by N-glycosylation, we performed genomewide expression profiling of yeast cells carrying a hypomorphic allele of ALG7. DNA microarrays showed that of more than 97% of known or predicted yeast genes, 29 displayed increased expression while 23 were repressed in alg7 mutants. Changes in transcript abundance were observed for a and alpha mating-type genes, for genes functioning in several mitogen-activated protein kinase (MAPK) cascades, as well as in phosphate, amino acid, carbohydrate, mitochondrial and ATP metabolism. Therefore, DNA microarrays have revealed direct and indirect targets, including internal feedback loops, through which N-glycosylation affects signaling and metabolic activities and is functionally linked with cellular regulatory circuitry.

Suppression of type 2 NO-synthase activity in macrophages by Candida albicans.

Macrophages (Mphi) are important for the defence against experimental disseminated candidiasis. Nitric oxide (NO) generated by the inducible isoform of NO-synthase (iNOS or NOS2) is thought to contribute to candidacidal effector functions by activated Mphi. In vitro, however, Mphi cannot control the growth and hyphal formation of Candida (C.) albicans. Using mouse peritoneal exudate Mphi stimulated with IFN-gamma and LPS, we examined the effect of C. albicans on NO synthesis, NOS2 enzyme activity and macrophage survival. C. albicans effectively inhibited the production of NO via suppression of total NOS2 protein and enzyme activity. Hyphal formation of C. albicans and direct interaction with host cells was required for maximum inhibition of NO production, whereas non-filamentous C. albicans mutants released soluble products that effected only partial inhibition. Ultimately, Mphi underwent apoptotic cell death after infection with C. albicans wild-type strains capable of hyphal formation, indicated by loss of the mitochondrial membrane potential and onset of chromatin degradation. NO suppression and Mphi killing are potent activities of C. albicans that may augment virulence of C. albicans.

Localization and signaling of G(beta) subunit Ste4p are controlled by a-factor receptor and the a-specific protein Asg7p.

Haploid yeast cells initiate pheromone signaling upon the binding of pheromone to its receptor and activation of the coupled G protein. A regulatory process termed receptor inhibition blocks pheromone signaling when the a-factor receptor is inappropriately expressed in MATa cells. Receptor inhibition blocks signaling by inhibiting the activity of the G protein beta subunit, Ste4p. To investigate how Ste4p activity is inhibited, its subcellular location was examined. In wild-type cells, alpha-factor treatment resulted in localization of Ste4p to the plasma membrane of mating projections. In cells expressing the a-factor receptor, alpha-factor treatment resulted in localization of Ste4p away from the plasma membrane to an internal compartment. An altered version of Ste4p that is largely insensitive to receptor inhibition retained its association with the membrane in cells expressing the a-factor receptor. The inhibitory function of the a-factor receptor required ASG7, an a-specific gene of previously unknown function. ASG7 RNA was induced by pheromone, consistent with increased inhibition as the pheromone response progresses. The a-factor receptor inhibited signaling in its liganded state, demonstrating that the receptor can block the signal that it initiates. ASG7 was required for the altered localization of Ste4p that occurs during receptor inhibition, and the subcellular location of Asg7p was consistent with its having a direct effect on Ste4p localization. These results demonstrate that Asg7p mediates a regulatory process that blocks signaling from a G protein beta subunit and causes its relocalization within the cell.

A conserved Gbeta binding (GBB) sequence motif in Ste20p/PAK family protein kinases.

Serine/threonine protein kinases of the Ste20p/PAK family are highly conserved from yeast to man. These protein kinases have been implicated in the signaling from heterotrimeric G proteins to mitogen-activated protein (MAP) kinase cascades and to cytoskeletal components such as myosin-I. In the yeast Saccharomyces cerevisiae, Ste20p is involved in transmitting the mating-pheromone signal from the betagamma-subunits of a heterotrimeric G protein to a downstream MAP kinase cascade. We have previously shown that binding of the G-protein beta-subunit (Gbeta) to a short binding site in the non-catalytic carboxy-terminal region of Ste20p is essential fortransmitting the pheromone signal. In this study, we searched protein sequence databases for sequences that are similar to the Gbeta binding site in Ste20p. We identified a sequence motif with the consensus sequence S S L phi P L I/V x phi phi beta (x: any residue; phi: A, I, L, S, or T; beta: basic residues) that is solely present in members of Ste20p/PAK family protein kinases. We propose that this sequence motif, which we have designated GBB (Gbeta binding) motif, is specifically responsible for binding of Gbeta to Ste20p/PAK protein kinases in response to activation of heterotrimeric G protein coupled receptors. Thus, the GBB motif is a novel type of signaling domain that serves to link protein kinases of the Ste20p/PAK family to G protein coupled receptors.

Molecular cloning of the CRM1 gene from Candida albicans.

In a screen for Candida albicans genes capable of supressing a ste20Delta mutation in Saccharomyces cerevisiae, a homologue of the exportin-encoding gene CRM1 was isolated. The CaCRM1 gene codes for a protein of 1079 amino acids with a predicted molecular weight of 124 029 and isoelectric point of 5.04. Crm1p from C. albicans displays significant amino acid sequence homology with Crm1p from Saccharomyces cerevisiae (65% identity, 74% similarity), Schizosaccharomyces pombe (55% identity, 66% similarity), Caenorhabditis elegans (45% identity, 57% similarity), and Homo sapiens (48% identity, 59% similarity). Interestingly, CaCRM1 encodes a threonine rather than a cysteine at position 533 in the conserved central region, suggesting that CaCrm1p is leptomycin B-insensitive, like S. cerevisiae Crm1p. CaCRM1 on a high copy vector can complement a thermosensitive allele of CRM1 (xpo1-1) in S. cerevisiae, showing that CaCrm1p and S. cerevisiae Crm1p are functionally conserved. Southern blot analysis suggests that CaCRM1 is present at a single locus within the C. albicans genome. The nucleotide sequence of the CaCRM1 gene has been deposited at GenBank under Accession No. AF178855.

A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex.

Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly.

Functional characterization of the interaction of Ste50p with Ste11p MAPKKK in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as the Schizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.

Cell cycle- and Cln2p-Cdc28p-dependent phosphorylation of the yeast Ste20p protein kinase.

Ste20p from Saccharomyces cerevisiae is a member of the Ste20/p21-activated protein kinase family of protein kinases. The Ste20p kinase is post-translationally modified by phosphorylation in a cell cycle-dependent manner, as judged by the appearance of phosphatase-sensitive species with reduced mobility on SDS-polyacrylamide gel electrophoresis. This modification is maximal during S phase, and correlates with the accumulation of Ste20p fused to green fluorescent protein at the site of bud emergence. Overexpression of Cln2p, but not Clb2p or Clb5p, causes a quantitative shift of Ste20p to the reduced mobility form, and this shift is dependent on Cdc28p activity. The post-translational mobility shift can be generated in vitro by incubation of Ste20p with immunoprecipitated Cln2p kinase complexes, but not by immunoprecipitated Clb2p or Clb5p kinase complexes. Ste20p is therefore a substrate for the Cdc28p kinase, and undergoes a Cln2p-Cdc28p mediated mobility shift as cells initiate budding and DNA replication. In cells that express only the Cln2p G1 cyclin, minor overexpression of Ste20p causes aberrant morphology, suggesting a proper coordination of Ste20p and Cln-Cdc28p activity may be required for the control of cell shape.

Characterization of Pak2p, a pleckstrin homology domain-containing, p21-activated protein kinase from fission yeast.

p21-activated kinases (PAKs) bind to and are activated by Rho family GTPases such as Cdc42 and Rac. Since these GTPases play key roles in regulating cell polarity, stress responses, and cell cycle progression, the ability of PAK to affect these processes has been examined. We previously showed that fission yeast pak1+ encodes an essential protein that affects mating and cell polarity. Here, we characterize a second pak gene (pak2+) from Schizosaccharomyces pombe. Like the Saccharomyces cerevisiae proteins Cla4p and Skm1p, fission yeast Pak2p contains an N-terminal pleckstrin homology domain in addition to a p21-binding domain and a protein kinase domain that are common to other members of the PAK family. Unlike pak1+, pak2(+) is not essential for vegetative growth or for mating in S. pombe. Overexpression of the wild-type pak2+ allele suppresses the lethal growth defect associated with deletion of pak1+, and this suppression requires both the pleckstrin homology- and the p21-binding domains of Pak2p, as well as kinase activity. A substantial fraction of Pak2p is associated with membranous components, an association mediated both by the pleckstrin homology- and by the p21-binding domains. These results show that S. pombe encodes at least two pak genes with distinct functions and suggest that the membrane localization of Pak2p, directed by its interactions with membrane lipids and Cdc42p, is critical to its biological activity.

Roles of the Candida albicans mitogen-activated protein kinase homolog, Cek1p, in hyphal development and systemic candidiasis.

Extracellular signal-regulated protein kinase (ERK, or mitogen-activated protein kinase [MAPK]) regulatory cascades in fungi turn on transcription factors that control developmental processes, stress responses, and cell wall integrity. CEK1 encodes a Candida albicans MAPK homolog (Cek1p), isolated by its ability to interfere with the Saccharomyces cerevisiae MAPK mating pathway. C. albicans cells with a deletion of the CEK1 gene are defective in shifting from a unicellular budding colonial growth mode to an agar-invasive hyphal growth mode when nutrients become limiting on solid medium with mannitol as a carbon source or on glucose when nitrogen is severely limited. The same phenotype is seen in C. albicans mutants in which the homologs (CST20, HST7, and CPH1) of the S. cerevisiae STE20, STE7, and STE12 genes are disrupted. In S. cerevisiae, the products of these genes function as part of a MAPK cascade required for mating and invasiveness of haploid cells and for pseudohyphal development of diploid cells. Epistasis studies revealed that the C. albicans CST20, HST7, CEK1, and CPH1 gene products lie in an equivalent, canonical, MAPK cascade. While Cek1p acts as part of the MAPK cascade involved in starvation-specific hyphal development, it may also play independent roles in C. albicans. In contrast to disruptions of the HST7 and CPH1 genes, disruption of the CEK1 gene adversely affects the growth of serum-induced mycelial colonies and attenuates virulence in a mouse model for systemic candidiasis.

Interaction of a G-protein beta-subunit with a conserved sequence in Ste20/PAK family protein kinases.

Serine/threonine protein kinases of the Ste20/PAK family have been implicated in the signalling from heterotrimeric G proteins to mitogen-activated protein (MAP) kinase cascades. In the yeast Saccharomyces cerevisiae, Ste20 is involved in transmitting the mating-pheromone signal from the betagamma-subunits (encoded by the STE4 and STE18 genes, respectively) of a heterotrimeric G protein to a downstream MAP kinase cascade. We have identified a binding site for the G-protein beta-subunit (Gbeta) in the non-catalytic carboxy-terminal regions of Ste20 and its mammalian homologues, the p21-activated protein kinases (PAKs). Association of Gbeta with this site in Ste20 was regulated by binding of pheromone to the receptor. Mutations in Gbeta and Ste20 that prevented this association blocked activation of the MAP kinase cascade. Considering the high degree of structural and functional conservation of Ste20/PAK family members and G-protein subunits, our results provide a possible model for a role of these kinases in Gbetagamma-mediated signal transduction in organisms ranging from yeast to mammals.

Virulence and hyphal formation of Candida albicans require the Ste20p-like protein kinase CaCla4p.

BACKGROUND: The pathogenic fungus Candida albicans is capable of a morphological transition from a unicellular budding yeast to a filamentous form. Extensive filamentous growth leads to the formation of mycelia displaying hyphae with branches and lateral buds. Hyphae have been observed to adhere to and invade host tissues more readily than the yeast form, suggesting that filamentous growth may contribute to the virulence of this major human pathogen. A molecular and genetic understanding of the potential role of morphological switching in the pathogenicity of C. albicans would be of significant benefit in view of the increasing incidence of candidiasis. RESULTS: The CaCLA4 gene of C. albicans was cloned by functional complementation of the growth defect of cells of the budding yeast Saccharomyces cerevisiae deleted for the STE20 gene and the CLA4 gene. CaCLA4 encodes a member of the Ste20p family of serine/threonine protein kinases and is characterized by a pleckstrin homology domain and a Cdc42p-binding domain in its amino-terminal non-catalytic region. Deletion of both alleles of CaCLA4 in C. albicans caused defects in hyphal formation in vitro, in both synthetic liquid and solid media, and in vivo in a mouse model for systemic candidiasis. The gene deletions reduced colonization of the kidneys in infected mice and suppressed C. albicans virulence in the mouse model. CONCLUSIONS: Our results demonstrate that the function of the CaCla4p protein kinase is essential for virulence and morphological switching of C. albicans in a mouse model. Thus, hyphal formation of C. albicans mediated by CaCla4p may contribute to the pathogenicity of this dimorphic fungus, suggesting that regulators of morphological switching may be useful targets for antifungal drugs.

Pheromone signalling and polarized morphogenesis in yeast.

Yeast cells respond to mating pheromones by activating a signal transduction pathway involving a seven transmembrane receptor/G protein complex linked to a mitogen-activated protein kinase module. Regulation of the G protein signal is controlled by the receptor and Sst2p; Sst2p may function as a GTPase-activating protein for the G protein alpha subunit. The Ste20 kinase acts in the linkage between the G protein and the MAP kinase module. Experiments suggest that binding of the Rho-like GTPase Cdc42p to Ste20p is not required for the mating response, yet is needed for the pseudohyphal growth response which involves many of the same kinases.

Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase.

Ste20p from Saccharomyces cerevisiae belongs to the Ste20p/p65PAK family of protein kinases which are highly conserved from yeast to man and regulate conserved mitogen-activated protein kinase pathways. Ste20p fulfills multiple roles in pheromone signaling, morphological switching and vegetative growth and binds Cdc42p, a Rho-like small GTP binding protein required for polarized morphogenesis. We have analyzed the functional consequences of mutations that prevent binding of Cdc42p to Ste20p. The complete amino-terminal, non-catalytic half of Ste20p, including the conserved Cdc42p binding domain, was dispensable for heterotrimeric G-protein-mediated pheromone signaling. However, the Cdc42p binding domain was necessary for filamentous growth in response to nitrogen starvation and for an essential function that Ste20p shares with its isoform Cla4p during vegetative growth. Moreover, the Cdc42p binding domain was required for cell-cell adhesion during conjugation. Subcellular localization of wild-type and mutant Ste20p fused to green fluorescent protein showed that the Cdc42p binding domain is needed to direct localization of Ste20p to regions of polarized growth. These results suggest that Ste20p is regulated in different developmental pathways by different mechanisms which involve heterotrimeric and small GTP binding proteins.

The phosphorylation site for Ste20p-like protein kinases is essential for the function of myosin-I in yeast.

The budding yeast Saccharomyces cerevisiae has two functionally redundant myosin-I isoforms encoded by the MYO3 and MYO5 genes. The function shared by these myosin proteins is required for proper yeast budding. Serine residue 357 in the head domain of Myo3p, conserved among myosin-I proteins including yeast Myo5p, was identified as a unique phosphorylation site for the serine/threonine protein kinase Ste20p and its closely related isoform Cla4p. These protein kinases share a function that is also essential for budding. Replacement of serine 357 with alanine disrupted the in vivo function of Myo3p, whereas this function was maintained by changing the serine residue to aspartate. This mutant version failed to compensate the growth defect of cells which lack both Ste20p and Cla4p, suggesting that myosin-I is not the only essential target of these protein kinases. Our results suggest that phosphorylation of the head domain by Ste20p-like protein kinases plays an essential role in the function of myosin-I in yeast cells.

Activation of myosin-I by members of the Ste20p protein kinase family.

The heavy chain of myosin-ID isolated from Dictyostelium was identified as an in vitro substrate for members of the Ste20p family of serine/threonine protein kinases which are thought to regulate conserved mitogen-activated protein kinase pathways. Yeast Ste20p and Cla4p and mammalian p21-activated protein kinase (PAK) phosphorylated the heavy chain to 0.5-0.6 mol of Pi/mol and stimulated the actin-dependent Mg2+-ATPase activity to an extent equivalent to that of the Ste20p-like myosin-I heavy chain kinase isolated from Dictyostelium. PAK purified from rat brain required GTPgammaS-Cdc42 to express full activity, whereas recombinant mouse mPAK3 fused to glutathione S-transferase and purified from bacteria, and Ste20p and Cla4p purified from yeast extracts were fully active without GTPgammaS-Cdc42. These results suggest, together with the high degree of structural and functional conservation of Ste20p family members and myosin-I isoforms, that myosin-I activation by Ste20p family protein kinases may contribute to the regulation of morphogenetic processes in organisms ranging from yeast to mammalian cells.