A role for the Pkc1p/Mpk1p kinase cascade in the morphogenesis checkpoint.

In many cells the timing of entry into mitosis is controlled by the balance between the activity of inhibitory Wee1-related kinases (Swe1p in budding yeast) and the opposing effect of Cdc25-related phosphatases (Mih1p in budding yeast) that act on the cyclin-dependent kinase Cdc2 (Cdc28p in budding yeast). Wee1 and Cdc25 are key elements in the G2 arrest mediated by diverse checkpoint controls. In budding yeast, a 'morphogenesis checkpoint' that involves Swe1p and Mih1p delays mitotic activation of Cdc28p. Many environmental stresses (such as shifts in temperature or osmolarity) provoke transient depolarization of the actin cytoskeleton, during which bud construction is delayed while cells adapt to environmental conditions. During this delay, the morphogenesis checkpoint halts the cell cycle in G2 phase until actin can repolarize and complete bud construction, thus preventing the generation of binucleate cells. A similar G2 delay can be triggered by mutations or drugs that specifically impair actin organization, indicating that it is probably actin disorganization itself, rather than specific environmental stresses, that triggers the delay. The G2 delay involves stabilization of Swe1p in response to various actin perturbations, although this alone is insufficient to produce a long G2 delay.

Phosphorylation-independent inhibition of Cdc28p by the tyrosine kinase Swe1p in the morphogenesis checkpoint.

The morphogenesis checkpoint in budding yeast delays cell cycle progression in G(2) when the actin cytoskeleton is perturbed, providing time for cells to complete bud formation prior to mitosis. Checkpoint-induced G(2) arrest involves the inhibition of the master cell cycle regulatory cyclin-dependent kinase, Cdc28p, by the Wee1 family kinase Swe1p. Results of experiments using a nonphosphorylatable CDC28(Y19F) allele suggested that the checkpoint stimulated two inhibitory pathways, one that promoted phosphorylation at tyrosine 19 (Y19) and a poorly characterized second pathway that did not require Cdc28p Y19 phosphorylation. We present the results from a genetic screen for checkpoint-defective mutants that led to the repeated isolation of the dominant CDC28(E12K) allele that is resistant to Swe1p-mediated inhibition. Comparison of this allele with the nonphosphorylatable CDC28(Y19F) allele suggested that Swe1p is still able to inhibit CDC28(Y19F) in a phosphorylation-independent manner and that both the Y19 phosphorylation-dependent and -independent checkpoint pathways in fact reflect Swe1p inhibition of Cdc28p. Remarkably, we found that a Swe1p mutant lacking catalytic activity could significantly delay the cell cycle in vivo during a physiological checkpoint response, even when expressed at single copy. The finding that a Wee1 family kinase expressed at physiological levels can inhibit a nonphosphorylatable cyclin-dependent kinase has broad implications for many checkpoint studies using such mutants in other organisms.

The morphogenesis checkpoint in Saccharomyces cerevisiae: cell cycle control of Swe1p degradation by Hsl1p and Hsl7p.

In Saccharomyces cerevisiae, the Wee1 family kinase Swe1p is normally stable during G(1) and S phases but is unstable during G(2) and M phases due to ubiquitination and subsequent degradation. However, perturbations of the actin cytoskeleton lead to a stabilization and accumulation of Swe1p. This response constitutes part of a morphogenesis checkpoint that couples cell cycle progression to proper bud formation, but the basis for the regulation of Swe1p degradation by the morphogenesis checkpoint remains unknown. Previous studies have identified a protein kinase, Hsl1p, and a phylogenetically conserved protein of unknown function, Hsl7p, as putative negative regulators of Swe1p. We report here that Hsl1p and Hsl7p act in concert to target Swe1p for degradation. Both proteins are required for Swe1p degradation during the unperturbed cell cycle, and excess Hsl1p accelerates Swe1p degradation in the G(2)-M phase. Hsl1p accumulates periodically during the cell cycle and promotes the periodic phosphorylation of Hsl7p. Hsl7p can be detected in a complex with Swe1p in cell lysates, and the overexpression of Hsl7p or Hsl1p produces an effective override of the G(2) arrest imposed by the morphogenesis checkpoint. These findings suggest that Hsl1p and Hsl7p interact directly with Swe1p to promote its recognition by the ubiquitination complex, leading ultimately to its destruction.

2-Bromopalmitoyl-CoA and 2-bromopalmitate: promiscuous inhibitors of membrane-bound enzymes.

2-Bromopalmitate and 2-bromopalmitoyl-CoA have been shown to inhibit a variety of enzymes and proteins associated with lipid metabolism. We found that both of the brominated compounds were non-competitive inhibitors of two microsomal activities of triacylglycerol biosynthesis, the mono- and diacylglycerol acyltransferases. With both compounds, the calculated Ki values were lower than the Km value for the palmitoyl-CoA substrate. In addition to inhibiting two other lipid synthetic activities, fatty acid CoA ligase and glycerol-3-P acyltransferase, 2-bromopalmitate and 2-bromopalmitoyl-CoA also inhibited two microsomal enzyme activities that are not related to lipid metabolism, NADPH cytochrome-c reductase and glucose-6-phosphatase. Inhibition of the three acyltransferases and fatty acid CoA ligase could be overcome by the addition of phospholipid vesicles, and 2-bromo[14C]palmitate readily labeled a large number of membrane-bound proteins as well as cytosolic proteins that had been solubilized in SDS. Thus, it appears likely that the inhibitory properties of the brominated compounds strongly depend on the effective concentration of the inhibitor within membranes rather than on any specific affinity for an acyl-chain binding region of the enzyme.

Protein kinase C-dependent regulation of human erythroleukemia (HEL) cell sphingosine kinase activity.

Sphingosine kinase functions in both the catabolism of sphingosine and in signal transduction pathways utilizing sphingosine-1-phosphate. The regulation of sphingosine kinase activity in human erythroleukemia (HEL) cells was investigated by treatment with several bioactive agents. Treatment of HEL cells with phorbol 12-myristate 13-acetate (PMA) caused a time- and concentration-dependent increase in sphingosine kinase activity measured in vitro. Sphingosine kinase activity increased in a phorbol ester- and diacylglycerol-specific manner. Staurosporine and calphostin C, protein kinase C (PKC) inhibitors, blocked the increased in sphingosine kinase activity, suggesting a PKC-dependent regulation. The effects of PMA on sphingosine kinase were dependent on transcription and translation. Purified PKC had no direct effect on sphingosine kinase activity. However, these studies led to the observation that HEL cell sphingosine kinase activity is stimulated in vitro by phosphatidylserine. Interestingly, other inducers of HEL cell differentiation, dimethylsulfoxide and retinoic acid, did not affect sphingosine kinase activity. These results indicate a separate and distinct pathway of PKC-dependent sphingosine kinase activation, and suggest a role for sphingosine kinase in regulation of cell function.

Perinatal hepatocyte/hepatoma hybrids: construction of clones that express the developmentally regulated monoacyglycerol acyltransferase activity.

Microsomal monoacyglycerol acyltransferase is a developmentally expressed enzyme that catalyzes the synthesis of sn-1,2-diacylglycerol from sn-2-monoacylglycerol and palmitoyl-CoA. The activity is present in liver from fetal and suckling rats but is absent in the adult. In order to obtain a stable permanent cell line that expresses this activity, Fao rat hepatoma cells and hepatocytes from 8-day-old baby rats were hybridized and clones were selected. Two hybrids (HA1 and HA7) expressed monoacylglycerol acyltransferase activity. Like fetal hepatocytes, but unlike hepatocytes from postnatal rats, the HA cells had high rates of [14C]acetate incorporation into glycerolipids, cholesterol, and cholesteryl esters, and they secreted triacylglycerol into the media. Monoacylglycerol acyltransferase specific activity increased 2.5-fold as the cells divided in culture, suggesting growth-dependent regulation. The specific activities of glycerol-P acyltransferase, the committed step of the microsomal pathway of glycerolipid synthesis, and diacylglycerol acyltransferase, the activity unique to triacylglycerol biosynthesis, were comparable to the levels of the corresponding activities in fetal hepatocytes. Addition of insulin or dexamethasone to the media increased the incorporation of [14C]oleate into triacyglycerol about 1.7-fold within 2 h, but had little effect on [14C]oleate incorporation into phospholipid. These hormonally responsive rat-hepatoma/hepatocyte hybrids reflect the fetal stage of hepatocyte development in five major aspects of lipid metabolism: sterol, fatty acid, and triacylglycerol biosynthesis, glycerolipid secretion, and the presence of the developmentally expressed monoacylglycerol pathway.

Solubilization and partial purification of neonatally expressed rat hepatic microsomal monoacylglycerol acyltransferase.

Rat hepatic microsomal monoacylglycerol acyltransferase (MGAT) is a developmentally expressed enzyme that catalyzes the formation of sn-1,2-diacylglycerol from sn-2-monoacylglycerol and fatty acyl-CoA. Treatment of suckling rat liver microsomes with various detergents showed that 0.3% Triton X-100, a nonionic detergent, solubilized a maximum amount of both protein (66%) and MGAT activity (56%). After solubilization with Triton X-100, MGAT was then purified 205-fold by sequential chromatography on QAE-Sephadex, CM-Sepharose (Fast Flow), and hydroxylapatite. Addition of phospholipids to the reaction mixture stimulated the purified enzyme activity more than 1.8-fold. sn-1,2-DiC18: 1-glycerol activated purified MGAT activity. Purified MGAT activity was specific for sn-2-monoacylglycerol; the activity with rac-1-monoC18:1-glycerol and rac-1- and sn-2-monoC18:1-glycerol ethers was less than 4% of the activity with sn-2-monoC18:1-glycerol. The purified MGAT had an isoelectric point of 9.7. The apparent Km and Vmax values of the purified enzyme for sn-2-monoC18:1-glycerol were 21 microM and 1036 nmol/min/mg, respectively. The apparent Km value for palmitoyl-CoA was 6.5 microM. Purified MGAT activity acylated sn-2-monoC18:2-glycerol and sn-2-monoC18:3-glycerol in preference to sn-2-monoC18:1-glycerol, consistent with a role for the monoacylglycerol pathway in retaining essential fatty acids.

Control of Swe1p degradation by the morphogenesis checkpoint.

In the budding yeast Saccharomyces cerevisiae, a cell cycle checkpoint coordinates mitosis with bud formation. Perturbations that transiently depolarize the actin cytoskeleton cause delays in bud formation, and a 'morphogenesis checkpoint' detects the actin perturbation and imposes a G2 delay through inhibition of the cyclin-dependent kinase, Cdc28p. The tyrosine kinase Swe1p, homologous to wee1 in fission yeast, is required for the checkpoint-mediated G2 delay. In this report, we show that Swe1p stability is regulated both during the normal cell cycle and in response to the checkpoint. Swe1p is stable during G1 and accumulates to a peak at the end of S phase or in early G2, when it becomes unstable and is degraded rapidly. Destabilization of Swe1p in G2 and M phase depends on the activity of Cdc28p in complexes with B-type cyclins. Several different perturbations of actin organization all prevent Swe1p degradation, leading to the persistence or further accumulation of Swe1p, and cell cycle delay in G2.

A phorbol ester binding domain of protein kinase C gamma. High affinity binding to a glutathione-S-transferase/Cys2 fusion protein.

Cysteine-rich regions of protein kinase C (PKC) are implicated in diacylglycerol-dependent regulation of kinase activity. The second cysteine-rich region (residues 92-173) of PKC gamma was expressed as a fusion protein with glutathione-S-transferase in Escherichia coli and purified to homogeneity by affinity chromatography. This fusion protein displayed high affinity phorbol dibutyrate (PDBu) binding (Kd 23 nM). The phosphatidylserine dependence of PDBu binding was highly cooperative with Hill numbers (near 4.5) similar to those previously reported for PKC gamma (Burns, D. J., and Bell, R. M. (1991) J. Biol. Chem. 266, 18330-18338). The fusion protein specifically bound 4 beta-hydroxy-PDBu but not the 4 alpha-stereoisomer. Furthermore, sn-1,2-dioctanoylglycerol (diC8) stereoselectively competed for PDBu binding. The cysteine-rich region was sufficient for association of the fusion protein to liposome preparations containing phosphatidylserine and phosphatidylcholine. Association was significantly enhanced in a stereospecific manner by the presence of PDBu as well as diC8. These results establish that a single cysteine-rich domain (residues 92-173) of PKC gamma contains regions necessary and sufficient for lipid-dependent stereospecific interactions with PDBu and diC8. Furthermore, the region is sufficient to confer translocation of a fusion protein to liposomes in a PDBu- and diC8-dependent fashion. Thus, a single cysteine-rich region of PKC gamma displays many of the properties characteristic of PKC.

A phorbol ester binding domain of protein kinase C gamma. Deletion analysis of the Cys2 domain defines a minimal 43-amino acid peptide.

Cysteine-rich regions of protein kinase C (PKC) are critical for the lipid-dependent regulation of activity and are implicated in the coordination of zinc. A glutathione S-transferase fusion protein containing the second cysteine-rich region, Cys2, of PKC gamma with bound zinc with a stoichiometry of 1.8 +/- 0.1 mol of zinc/mol of protein. Deletion analysis within this cysteine-rich region defined amino acids essential for zinc coordination. An NH2-terminal histidine (His102) and a COOH-terminal cysteine (Cys151) were both critical for the coordination of distinct zinc atoms. Both represent the ultimate residues of a 50-amino acid consensus motif with six conserved cysteines and two conserved histidines present in the cysteine-rich regions of all PKC isoforms. Removal of histidine His102 abolished phorbol ester binding, while deletion of cysteine Cys151 did not. Deletion of valine (Val147) greatly diminished phorbol ester binding, which was completely lost only when valine (Val144) was also deleted. No significant further reduction in zinc stoichiometry below one resulted even when three COOH-terminal conserved cysteines (Cys151, Cys143, and Cys135) and a conserved histidine (His140) were deleted. These results are consistent with a model in which two zinc atoms are tetracoordinated per cysteine-rich region in two independent coordination spheres that are not functionally equivalent. These analyses determine a minimal peptide (residues 102-144) of 43 amino acids capable of [3H]PDBu binding.

Combinatorial control of cyclin B1 nuclear trafficking through phosphorylation at multiple sites.

Entry into mitosis is regulated by the Cdc2 kinase complexed to B-type cyclins. We and others recently reported that cyclin B1/Cdc2 complexes, which appear to be constitutively cytoplasmic during interphase, actually shuttle continually into and out of the nucleus, with the rate of nuclear export exceeding the import rate (). At the time of entry into mitosis, the import rate is increased, whereas the export rate is decreased, leading to rapid nuclear accumulation of Cdc2/cyclin B1. Although it has recently been reported that phosphorylation of 4 serines within cyclin B1 promotes the rapid nuclear translocation of Cdc2/cyclin B1 at G(2)/M, the role that individual phosphorylation sites play in this process has not been examined (, ). We report here that phosphorylation of a single serine residue (Ser(113) of Xenopus cyclin B1) abrogates nuclear export of cyclin B1. This serine lies directly within the cyclin B1 nuclear export sequence and, when phosphorylated, prevents binding of the nuclear export factor, CRM1. In contrast, analysis of phosphorylation site mutants suggests that coordinate phosphorylation of all 4 serines (94, 96, 101, and 113) is required for the accelerated nuclear import of cyclin B1/Cdc2 characteristic of G(2)/M. Additionally, binding of cyclin B1 to importin-beta, the factor known to be responsible for the slow interphase nuclear entry of cyclin B1, appears to be unaffected by the phosphorylation state of cyclin B. These data suggest that a distinct import factor must be recruited to enhance nuclear entry of Cdc2/cyclin B1 at the G(2)/M transition.

The regulatory domain of protein kinase C coordinates four atoms of zinc.

Protein kinase C (PKC) was found to be a zinc metallo-enzyme. Atomic absorption measurements on the intact enzyme indicated that four zinc atoms (4.2 +/- 0.5) were bound per PKC alpha molecule. Similar stoichiometric ratios were determined for PKC beta II and PKC gamma, other PKC isoforms individually expressed in the baculovirus-insect cell expression system, as well as for purified rat brain PKC. By trypsin treatment of PKC alpha, a 32-kDa lipid binding regulatory and a 50-kDa catalytic domain were generated that were subsequently completely separated by gel filtration in the presence of Triton X-100/phosphatidylserine mixed micelles. Zinc was present at levels significantly above background in fractions that contained the 32-kDa fragment and displayed phorbol ester binding activity. Lipid association and phorbol ester binding did not lead to displacement of zinc from the protein. The stoichiometry determined for this fragment (4.7 +/- 0.9) suggested that zinc was bound exclusively within the lipid binding regulatory domain of intact PKC. Furthermore, this stoichiometry is consistent with zinc being coordinated between 6 cysteine residues in a structural motif related to the Zn(II)2Cys6 binuclear cluster identified in the GAL4 transcriptional factor (Pan, T., and Coleman, J.E. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2077-2081).

Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p.

In budding yeast cells, the cytoskeletal polarization and depolarization events that shape the bud are triggered at specific times during the cell cycle by the cyclin-dependent kinase Cdc28p. Polarity establishment also requires the small GTPase Cdc42p and its exchange factor, Cdc24p, but the mechanism whereby Cdc28p induces Cdc42p-dependent polarization is unknown. Here we show that Cdc24p becomes phosphorylated in a cell cycle-dependent manner, triggered by Cdc28p. However, the role of Cdc28p is indirect, and the phosphorylation appears to be catalyzed by the p21-activated kinase family member Cla4p and also depends on Cdc42p and the scaffold protein Bem1p. Expression of GTP-Cdc42p, the product of Cdc24p-mediated GDP/GTP exchange, stimulated Cdc24p phosphorylation independent of cell cycle cues, raising the possibility that the phosphorylation is part of a feedback regulatory pathway. Bem1p binds directly to Cdc24p, to Cla4p, and to GTP-bound Cdc42p and can mediate complex formation between these proteins in vitro. We suggest that Bem1p acts to concentrate polarity establishment proteins at a discrete site, facilitating polarization and promoting Cdc24p phosphorylation at specific times during the cell cycle.

Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1.

Activation of the Cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Cyclin B1 localization changes dramatically during the cell cycle, precipitously transiting from the cytoplasm to the nucleus at the beginning of mitosis. Presumably, this relocalization promotes the phosphorylation of nuclear targets critical for chromatin condensation and nuclear envelope breakdown. We show here that the previously characterized cytoplasmic retention sequence of Cyclin B1, responsible for its interphase cytoplasmic localization, is actually an autonomous nuclear export sequence, capable of directing nuclear export of a heterologous protein, and able to bind specifically to the recently identified export mediator, CRM1. We propose that the observed cytoplasmic localization of Cyclin B1 during interphase reflects the equilibrium between ongoing nuclear import and rapid CRM1-mediated export. In support of this hypothesis, we found that treatment of cells with leptomycin B, which disrupted Cyclin B1-CRM1 interactions, led to a marked nuclear accumulation of Cyclin B1. In mitosis, Cyclin B1 undergoes phosphorylation at several sites, a subset of which have been proposed to play a role in Cyclin B1 accumulation in the nucleus. Both CRM1 binding and the ability to direct nuclear export were affected by mutation of these phosphorylation sites; thus, we propose that Cyclin B1 phosphorylation at the G2/M transition prevents its interaction with CRM1, thereby reducing nuclear export and facilitating nuclear accumulation.