Mammalian Cdk5 is a functional homologue of the budding yeast Pho85 cyclin-dependent protein kinase Dongqing Huang*, Gentry Patrick†, Jason Moffat*, Li-Huei Tsai†, and Brenda Andrews*‡ *Department of Molecular and Medical Genetics, University of Toronto, 1 Kings College Circle, Toronto, Canada M5S 1A8; and †Howard Hughes Medical Institute and Department of Pathology, Harvard Medical School, Boston, MA 02115 Edited by Ira Herskowitz, University of California, San Francisco, CA, and approved October 13, 1999 (received for review May 26, 1999) Mammalian Cdk5 is a member of the cyclin-dependent kinase display a broad spectrum of phenotypes, including slow growth family that is activated by a neuron-specific regulator, p35, to in rich medium, derepressed acid phosphatase activity, hyper- ͞ regulate neuronal migration and neurite outgrowth. p35 Cdk5 accumulation of glycogen, and defects in G1 progression and kinase colocalizes with and regulates the activity of the Pak1 kinase actin cytoskeleton regulation (11–15). Consistent with the mul- in neuronal growth cones and likely impacts on actin cytoskeletal tifunctional nature of Pho85, 10 genes encoding known or dynamics through Pak1. Here, we describe a functional homologue putative Pho85 cyclins (Pcls) have been identified (16). Expres- of Cdk5 in budding yeast, Pho85. Like Cdk5, Pho85 has been sion of three of the PCLs—PCL1, PCL2, and PCL9—is cell implicated in actin cytoskeleton regulation through phosphoryla- cycle-regulated with peak transcript levels in G1 phase, whereas tion of an actin-regulatory protein. Overexpression of CDK5 in expression of other Pho85 cyclins is constant through the cell yeast cells complemented most phenotypes associated with cycle. pho85⌬, including defects in the repression of acid phosphatase Like Cdk5, Pho85 plays important roles in polarized cell expression, sensitivity to salt, and a G1 progression defect. Con- growth and cytoskeletal dynamics. Yeast strains lacking PCL1,2- sistent with the functional complementation, Cdk5 associated with like cyclins are morphologically abnormal and display pheno- and was activated by the Pho85 cyclins Pho80 and Pcl2 in yeast types consistent with a disruption of the actin cytoskeleton (11, cells. In a reciprocal series of experiments, we found that Pho85 16, 17). Pcl-Pho85 kinases contribute to actin regulation at least associated with the Cdk5 activators p35 and p25 to form an active in part through phosphorylation of the Rvs167 protein (11). kinase complex in mammalian and insect cells, supporting our Rvs167 is a modular protein composed of at least three distinct hypothesis that Pho85 and Cdk5 are functionally related. Our protein-interaction domains and likely functions in multiprotein results suggest the existence of a functionally conserved pathway complexes to regulate actin polarization (17, 18). Among Cdks, involving Cdks and actin-regulatory proteins that promotes reor- Pho85 is most closely related to Cdk5, with 56% identity and ganization of the actin cytoskeleton in response to regulatory 72% similarity at the primary amino acid level. To explore the signals. functional significance of the protein sequence similarity be- tween Pho85 and Cdk5, and their similar roles in the regulation yclin-dependent kinases (Cdks) comprise a large family of of asymmetric growth, we used in vivo complementation assays Cproteins whose members are well known as key regulators and in vitro reconstitution to show that Pho85 and Cdk5 are that promote cell cycle progression in eukaryotic cells (reviewed functional homologues. Our observations led us to propose the in ref. 1). Recent studies have highlighted roles for Cdks in existence of a functionally conserved regulatory pathway, in- postmitotic cells, in gene expression, and in cell regulation volving cyclin-dependent kinases and their targets, that regulates beyond cell cycle control (2, 3). Although highly homologous to the actin cytoskeleton in response to regulatory stimuli. other members of the Cdk family, mammalian Cdk5 has no Materials and Methods known function in proliferative cells. Rather, Cdk5-associated Yeast Strains and Media. The wild-type (BY263, MATa trp1⌬63 kinase activity is restricted to postmitotic neurons and Cdk5 is ⌬ ⌬ ⌬ required for histogenesis of the central nervous system and leu2- 1 his3 200 ura3–52 lys2 ade2) and pho85 strains (BY391, neurite outgrowth (2, 4–6). Cdk5 phosphorylates syntaxin 1, pho85::LEU2, otherwise isogenic to BY263) have been de- Munc18, and synapsin I (7, 8), suggesting a regulatory role for scribed (16). We used a PCR-based strategy to target a triple- actin cytoskeletal dynamics. In the growth cones of cultured hemagglutinin (HA) epitope tag to the N-terminal coding cortical neurons, Cdk5 and its neuron-specific activator, p35, sequence of PCL2 in strain BY263. PCR conditions were as colocalize with actin filaments at the growth cone periphery (5). described by using plasmid pFA6a-kanMX6-PGAL1–3HA as More recently, p35͞Cdk5 was shown to colocalize with two template (19). The epitope-tagged allele of PCL2 is express- actin-regulatory proteins, the Rho-type GTPase Rac and the ed from the inducible GAL promoter (strain 263HA- PCL2, BY1116, MATa trp1 leu2 his3 ura3 lys2 ade2 Pak1 kinase, in neuronal growth cones (9). Phosphorylation of r Pak1 kinase by p35-Cdk5 may contribute to the reorganization pcl2::pGAL-HA-PCL2::Kan ). A pho85 deletion strain carrying of the actin cytoskeleton that occurs during neurite outgrowth the tagged PCL2 allele (pho85HA-PCL2, BY1129) was con- structed by crossing strain 263HA-PCL2 to strain BY864 and neuronal migration. ⌬ In the budding yeast Saccharomyces cerevisiae, two Cdk com- [pho85 TRP1 (11)]. Strain BY794 was used to assay comple- plexes, Cln-Cdc28 and Pcl-Pho85, have been implicated in the mentation of the G1 arrest phenotype in a pho85 cln1 cln2 mutant and had the following genotype: cln1⌬TRP1 cln2⌬LEU2 GENETICS regulation of the actin cytoskeleton. The Cdc28 kinase is re- ⌬ sponsible for cell cycle transitions and can be activated by nine pho85 LEU2 (pMETpho85ts17-HIS3), otherwise isogenic to cyclin regulatory subunits, the Clns and the Clbs (reviewed in ref. 3). Inappropriate expression of Cln-Cdc28 kinases leads to This paper was submitted directly (Track II) to the PNAS office. altered bud emergence, suggesting a role for Cdc28 in regulating Abbreviations: Cdk, cyclin-dependent kinase; Pcl, Pho85 cyclin; HA, hemagglutinin; GST, cell polarity and budding (10). The Pho85 Cdk is also activated glutathione S-transferase. by multiple cyclins and has roles in both cell cycle control and ‡To whom reprint requests should be addressed. E-mail: [email protected]. metabolic processes (3). Although PHO85 is not an essential The publication costs of this article were defrayed in part by page charge payment. This gene, it is required for viability in some stress conditions, such article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. as growth after starvation (11). In addition, pho85 mutants §1734 solely to indicate this fact. PNAS ͉ December 7, 1999 ͉ vol. 96 ͉ no. 25 ͉ 14445–14450 Downloaded by guest on September 29, 2021 BY263. A slt2⌬LEU2 pho85⌬TRP1 strain was constructed by mating strains Y782 [slt2⌬LEU2 (20)] and BY864 (pho85⌬TRP1). The resulting diploid strain was transformed with either plasmid pGALCDK5 (see below, pCDK5) or pGAL- PHO85-HIS3 (12), and pho85 slt2 double mutants were recov- ered by dissecting tetrads on galactose medium. Yeast cells were grown in supplemented minimal medium with either 2% dex- trose (SD) or galactose (SG). Low- and high-phosphate SG media were prepared by using phosphate-depleted yeast nitro- gen base and KH2PO4 supplements. Salt-containing medium was prepared by addition of NaCl to 0.75 M. COS-7 and Insect Cell Culture. COS-7 cells were maintained in DMEM supplemented with 10% calf serum and penicillin͞ streptomycin. Transient transfections of pCDNA3-based p35, p25, Cdk5, and PHO85 expression constructs in COS-7 cells were performed by using standard calcium phosphate methods. Hi5 insect cells were maintained in Grace’s Insect media and were infected with baculovirus 24 hr before harvesting for lysate preparation. Fig. 1. Biochemical and genetic complementation of the acid phosphatase Plasmids. To express PHO80 from the GPD promoter in yeast, an regulation defect in a pho85⌬ mutant by Cdk5 expression. (A) Complemen- HA tagged-PHO80 allele (21) was cloned into vector pBA230v tation of defect in acid phosphatase regulation. Acid phosphatase activity was (ATCC no. 87357, 2-␮, TRP1, GPD promoter) to create plasmid measured in pho85⌬ strains containing pCDK5 or pCDK5-I and a PHO80 pGPDPHO80 (pBA1276). Likewise, plasmid pBA230v-p35 expression plasmid (pPho80) or a control vector (p230v). Acid phosphatase (pBA1277) was constructed by subcloning the p35 gene from activity from a wild-type strain is also shown (wt). These strains were grown in plasmid pcDNA3-p35 (see below) into pBA230v. Vectors for repressed (7.4 mM phosphate, shaded bars) and derepressed (3.7 ␮M phos- expressing CDK5 (pMR438PSS) and antisense CDK5 (CDK5-I, phate, solid bars) conditions. Values are normalized for cell density (OD600). (B) pMR438SSP) from the GAL promoter in yeast have been Pho4 kinase assays with HA-Pho80-Cdk5. HA-Pho80 was immunoprecipitated described (22). A PHO85 expression plasmid was constructed by from yeast extracts and used in kinase assays with Pho4 as substrate. The ␮ following strains were used for extract preparation: lane