The yeast Saccharomyces cerevisiae contains two functional homologues of the ras oncogene family, RAS1 and RAS2. These genes are required for growth, and all evidence indicates that this essential function is the activation of adenylate cyclase. In contrast, ras in mammalian cells does not appear to influence adenylate cyclase activity. To clarify the relation between ras function in yeast and in higher eukaryotes, and the role played by yeast RAS in growth control, it is necessary to identify functions acting upstream of RAS in the adenylate cyclase pathway. The evidence presented here indicates that CDC25, identified by conditional cell cycle arrest mutations, encodes such an upstream function.

Activated versions of ras genes have been found in various types of malignant tumors. The normal versions of these genes are found in organisms as diverse as mammals and yeasts. Yeast cells that lack their functional ras genes, RASSC-1 and RASSC-2, are ordinarily nonviable. They have now been shown to remain viable if they carry a mammalian rasH gene. In addition, yeast-mammalian hybrid genes and a deletion mutant yeast RASSC-1 gene were shown to induce morphologic transformation of mouse NIH 3T3 cells when the genes had a point mutation analogous to one that increases the transforming activity of mammalian ras genes. The results establish the functional relevance of the yeast system to the genetics and biochemistry of cellular transformation induced by mammalian ras genes.

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types.In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCKI and YCK2 is a 60-to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-1B-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying theyck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2" allele and deletion of CDCSS indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.Casein kinase I (CK I) is the name given to a Ser/Thr protein kinase activity found in all eukaryotic cells. Extensive characterization of CK I activities from a number of cell types has revealed several distinguishing properties of these activities, including specificity for acidic substrates and independence of classical second messengers (reviewed in reference 41). Classical CK I phosphorylation sites have been defined as acidic amino acids upstream of a Ser or Thr residue (41). However, for one isoform, substrates containing the motif Ser/Thr(PO4)-X-X-Ser/Thr are recognized with high affinity (10-12), suggesting that a phosphorylated residue can provide a necessary recognition determinant. Such substrate specificity in vivo could link the activity of CK I enzymes with activities of other protein kinases in a synergistic hierarchical mechanism, in which a primary phosphorylation event can generate a recognition site for CK I (31). Thus, CK I activity could be recruited into second messenger-dependent phosphorylation cascades.CK I activities are found in multiple subcellular compartments (41). Nuclear, cytoplasmic, plasma membrane, and microsomal fractions of various cell types have been found to contain CK I activities. Although historically CK I activity has been associated with a single 34-to 37-kDa subunit, molecular sizes associated with CK I activity vary from 34 to 55 kDa between cell types and even between subcellular fractions (41). The hypothesis that these activities could correspond to discretely encoded isoforms has been confirmed by the recent isolation of multiple related cDNA clones from mammalian tissues. At least four closely related isoforms are encoded in bovine brain (37), and additional representatives of these isoforms have been identified subsequently in other vertebrate cell types (4, 16).Central roles for CK I phosphorylation in cellul...

We report the isolation of an essential pair of Saccharomyces cerevisiae genes that encode protein kinase homologues. The two genes were independently isolated as dosage-dependent suppressors. Increased dosage of YCKI suppressed defects caused by reduced SNF1 protein kinase activity, and increased dosage of YCK2 relieved sensitivity of wild-type cells to salt stress. The two genes function identically in the two growth assays, and loss of function of either gene alone has no discernible effect on growth. However, loss of function of both genes results in inviability. The (1); components of the translation apparatus including initiation factors 4B, 4E, and 5 and tRNA synthetases (1, 4, 5); and metabolic enzymes, notably glycogen synthase (1).The site preference of casein kinase I has been defined as acidic residues amino-terminal to the modified residue (1, 6). However, the sequence motif -Ser(P)-Xaa-Xaa-Ser-has recently been demonstrated to specify casein kinase I action, suggesting a model for casein kinase I biological activity termed hierarchical protein phosphorylation (7). For example, phosphorylation of N-CAM by casein kinase I in vitro is abolished by prior treatment with phosphatase (3). Also, phosphorylation of glycogen synthase by cAMP-dependent protein kinase (cAPK) potentiates that by casein kinase 1 (6). Replacement of phosphoserine by a block of three or four acidic residues in synthetic peptide substrates results in poor but relatively specific casein kinase I substrates (8).Casein kinase I activity has been inferred to be central to cellular function from its wide distribution, numerous in vitro substrates, and multiple subcellular localizations. However, the importance of phosphorylation by the enzyme in vivo has been demonstrated in few cases, notably glycogen synthase. Phosphorylation of glycogen synthase by casein kinase I in vitro results in inactivation of the enzyme (7) and phosphorylation of one site, Ser10, occurs in rabbit muscle in vivo in response to stimulation by epinephrine (9). Other physiologically important phosphorylations by casein kinase I have been suggested but its overall physiological roles remain unclear. The study of casein kinase I function would be greatly facilitated by the approaches that are possible with the genetically tractable budding yeast Saccharomyces cerevisiae. Enzymological studies have revealed multiple forms of casein kinase in yeast that share properties with mammalian casein kinase I (10-12), but no biological function has been assigned to these activities.We describe here the isolation of two structural homologues of casein kinase I in S. cerevisiae. The YCKI and YCK2 (yeast casein kinase I homologue) genes were identified independently based on the effects of increased gene dosage. YCKJ was isolated as a suppressor of the requirement for SNF4 function. The SNF4 protein is a positive effector ofthe SNF1 protein kinase (13), which is required for carbon catabolite derepression (14 28The publication costs of this article were defrayed in pa...

Saccharomyces cerevisiae contains two genes with remarkable homology to members of the ras oncogene family. These two genes, RAS] and RAS2, constitute an essential gene family since spores with disruptions ofboth genes fail to grow. We report here that strains containing RAS2 disruptions have three distinct phenotypes. First, they fail to grow efficiently on nonfermentable carbon sources. Like their mammalian counterparts, the yeast RAS proteins bind GTP (7, 8) and, at least in the case of RAS1, have an intrinsic GTPase activity similar to that found in p21 (9). This activity is decreased or absent in RAS1 proteins containing missense mutations at amino acid positions analogous to transforming mutations in p21 (9). Functional similarities also exist between p21 and yeast RAS. The normal mammalian Harvey ras gene (10) or the viral Harvey ras (11) can substitute for both of the normal RAS genes in yeast and a modified RAS] gene can transform NIH-3T3 cells (11).Given the functional and biochemical similarities between mammalian and yeast RAS proteins, it is likely that knowledge about RAS function in yeast may have direct implications forRAS function in mammals. We report here that yeast strains lacking RAS2 function have a defect in gluconeogenic growth, accumulate excessive levels of storage carbohydrates, and sporulate prematurely. We have identified mutations in three additional genes that suppress the gluconeogenic defect. Two of these mutations allow strains without RAS to grow. MATERIALS AND METHODSYeast Strains. TX2-530.1.1C (a leu2 ura3 trpl lysi lys2 his3 ras2-530) (4) was backcrossed to MCY638 (a his4-539 lys2-801 ura3-52). MCY638 and MCY317 (a ade2-101 his4-539), derived from strain S288C, were kindly provided by Marian Carlson. Meiotic segregants of EG81, the second backcross to MCY638, were used for quantitative measurements and revertant analysis. Although backcrosses were performed to show linkage between specific phenotypes and ras2, mendelian segregation patterns for the gluconeogenic defect and glycogen hyperaccumulation were clearly observed in original transformed diploid TX2-530. Specific strains used include EG81-40A (a ura3 his4 lys2), EG81-40B (a ras2-530 leu2 ura3 his4 lys2 lysi), EG81-40C (a ras2-530 leu2 ura3 his4 lys2 his3 lysl?), EG81-40D (a ura3 his4 lys2 his3 lysl?), , and 112-699 [a leu2 ura3 can1-100 ade2-1 his3 ras2-699 (His+)].The gluconeogenic revertants described below were derived from EG81-22B. Revertants were crossed to EG72-16C (a ras2-530 leu2 trpl lys2) and all subsequent crosses between the strains containing sra mutations were done with meiotic progeny from these crosses. EG73-14D (a rasl-545 leu2 ura3 lysi his3) and EG87-2B (a rasl-545 ura3 his4 lys2) were used to test if the sra mutations bypass RAS. XC0262485 [a rasl485 (Trp') stell'3 leu2 his4-580 his3 trpl] has been described elsewhere (7).Media and Genetic Techniques. Unless otherwise stated, yeast cells were grown on 1% yeast extract and 2% peptone (YEP medium) containing 2% of the designated carbon sourc...

Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABA A receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABA A receptors composed of either ␣ 1 ␤ 2 ␥ 2 L or ␣ 1 ␤ 2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor ␤ 2 subunit that is critical for endocytosis. Internalization of GABA A receptors lacking this motif is dramatically inhibited, and the receptors appear to accumulate on the cell surface. Patch clamp analysis of receptors lacking the dileucine motif show that there is an increase in the peak amplitude of GABA-gated chloride currents compared with wild-type receptors. Additionally, GABA-gated chloride currents in HEK 293 cells expressing wild-type receptors are increased by introduction of a peptide corresponding to the dileucine motif region of the receptor ␤ 2 subunit but not by a control peptide containing alanine substitutions for the dileucine motif. In mouse brain cerebral cortical neurons, the dileucine motif peptide increases GABA-gated chloride currents of native GABA A receptors. This is the first report to our knowledge that an AP2 adaptin dileucine recognition motif is critical for the endocytosis of ligand-gated ion channels belonging to this superfamily.The GABA A receptor is a ligand-gated chloride channel that, upon activation by GABA 1 (␥-aminobutyric acid), mediates increases in chloride conductance resulting in membrane hyperpolarization and neuronal inhibition (1). The role of these receptors in hyperexcitability states, such as epilepsy and anxiety, is widely recognized. Importantly, GABA A receptors mediate the effects of benzodiazepines and barbiturates, two frequently prescribed classes of therapeutic agents. The GABA A receptor is a pentameric receptor composed of multiple subunits, each containing four membrane-spanning regions (M1-M4) with a large intracellular loop between M3 and M4. A number of subunits exist (␣ 1Ϫ6 , ␤ 1Ϫ3 , ␥ 1Ϫ3 , ␦, , ⑀, ), and receptors composed of ␣ 1 ␤ 2 ␥ 2 L subunits are believed to represent the predominant GABA A receptor subtype in the brain (1).Receptor endocytosis is known to regulate the cell surface expression of neurotransmitter receptors, and such regulation is an important mechanism for controlling the synaptic efficacy of neurotransmitters (2). Although GABA A receptors undergo endocytosis, the mechanism is not well understood. Several lines of evidence indicate that GABA A receptor endocytosis may be clathrin/dynamin-dependent. These include the presence of GABA A receptors in clathrin-coated vesicles isolated from brain (3), the colocalization of the receptor with transferrin receptors (4), and the colocalization and co-immunoprecipitation of hippocampal GABA A receptors with the clathrin adaptor complex AP2 adaptin (5). Additionally, peptides that dis...

The inhibition of ␥-aminobutyric acid (GABA)-gated chloride currents by the protein kinase C (PKC) activator 4␤-phorbol 12-myristate 13-acetate (PMA) was investigated using recombinant human GABA A receptors expressed in Xenopus oocytes. PMA (5 nM) reduced the GABA response in oocytes expressing the ␣1␤2␥2L receptor construct, as measured by the two-electrode voltage-clamp method. GABA responses declined to approximately 25% of their pretreatment value within 45 min. GABA responses in oocytes expressing a receptor construct from which the known PKC phosphorylation sites were absent, ␣1␤2(S410A), were comparably inhibited. Phorbol 12-monomyristate (PMM; 5 nM), which does not activate PKC, did not alter the GABA response in either construct, while the PKC inhibitor calphostin C (0.5 M) prevented the PMA effect. To further investigate PMA inhibition of the GABA response, a GABA A receptor ␣1 subunit/green fluorescent protein (GFP) chimera (␣1GFP) was used to visualize GABA A receptor distribution. Similar to the wild type constructs, PMA robustly decreased GABA responses in oocytes expressing ␣1GFP␤2␥2L and ␣1GFP␤2(S410A) receptor constructs. Following PMA treatment, GFP fluorescence in the oocyte plasma membrane was decreased to approximately 45% of the pretreatment values indicating GABA A receptor internalization. This effect of PMA was prevented by calphostin C and was not produced by PMM. Experiments with bd24, a monoclonal antibody which recognizes an extracellular epitope of the ␣1 subunit, were used to demonstrate that PMA, but not PMM, decreases ␣1 subunit immunoreactivity in the plasma membrane of intact oocytes expressing the ␣1␤2␥2L construct, thus confirming the results obtained with the chimeric receptor. It is concluded that, in Xenopus oocytes, PMA induces an internalization of the GABA A receptor through PKC-mediated phosphorylation of an unidentified protein(s) and that this contributes to the decrease in electrophysiological responses to GABA following PKC activation. ␥-Aminobutyric acid (GABA)1 is the principal inhibitory neurotransmitter in the vertebrate central nervous system. The fast inhibitory actions of GABA are mediated by the GABA A receptor, a postsynaptic ligand-gated chloride channel. At least 17 receptor subunit subtypes have been identified (␣ 1-6 , ␤ 1-4 , ␥ 1-4 , ␦, 1,2 ), and the receptor is thought to form a heteropentamer (1). The subunit composition of the receptor determines agonist potency (2, 3), desensitization kinetics (3), phosphorylation (4 -6), and allosteric properties (2, 7, 8).Calcium-phospholipid-dependent protein kinase (PKC) phosphorylates purified GABA A receptors on polypeptides corresponding to ␤ subunits (9), and PKC phosphorylation sites have been identified on a variety of GABA A subunits including ␤1 serine 409 (5, 6), ␥2S serine 327, and ␥2L serine 343/327 (4, 5). Activation of PKC by phorbol esters such as 4␤-phorbol 12-myristate 13-acetate (PMA) inhibits GABA A receptor function in mouse brain cerebellar microsacs (10), cultured cervical ganglion neuro...

Casein kinase 1 protein kinases are ubiquitous and abundant Ser/Thr-specific protein kinases with activity on acidic substrates. In yeast, the products of the redundant YCK1 andYCK2 genes are together essential for cell viability. Mutants deficient for these proteins display defects in cellular morphogenesis, cytokinesis, and endocytosis. Yck1p and Yck2p are peripheral plasma membrane proteins, and we report here that the localization of Yck2p within the membrane is dynamic through the cell cycle. Using a functional green fluorescent protein (GFP) fusion, we have observed that Yck2p is concentrated at sites of polarized growth during bud morphogenesis. At cytokinesis, GFP–Yck2p becomes associated with a ring at the bud neck and then appears as a patch of fluorescence, apparently coincident with the dividing membranes. The bud neck association of Yck2p at cytokinesis does not require an intact septin ring, and septin assembly is altered in a Yck-deficient mutant. The sites of GFP–Yck2p concentration and the defects observed for Yck-deficient cells together suggest that Yck plays distinct roles in morphogenesis and cytokinesis that are effected by differential localization.