Ammonium permease-based sensing mechanism for rapid ammonium activation of the protein kinase A pathway in yeast

In the yeast Saccharomyces cerevisiae starvation for nitrogen on a glucose-containing medium causes entrance into G0 and downregulation of all targets of the PKA pathway. Re-addition of a nitrogen source in the presence of glucose causes rapid activation of trehalase and other PKA targets. Trehalase activation upon ammonium re-supplementation is dependent on PKA activity, but not on its regulatory subunit nor is it associated with an increase in cAMP. In nitrogen-starved cells, ammonium transport and activation of trehalase are most active in strains expressing either the Mep2 or Mep1 ammonium permease, as opposed to Mep3. The non-metabolizable ammonium analogue, methylamine, also triggers activation of trehalase when transported by Mep2 but not when taken up by diffusion. Inhibition of ammonium incorporation into metabolism did not prevent signalling. Extensive site-directed mutagenesis of Mep2 showed that transport and signalling were generally affected in a similar way, although they could be separated partially by specific mutations. Our results suggest an ammonium permease-based sensing mechanism for rapid activation of the PKA pathway. Mutagenesis of Asn246 to Ala in Mep2 abolished transport and signalling with methylamine but had no effect with ammonium. The plant AtAmt1;1, AtAmt1;2, AtAmt1;3 and AtAmt2 ammonium transporters sustained transport and trehalase activation to different extents. Specific mutations in Mep2 affected the activation of trehalase differently from induction of pseudohyphal differentiation. We also show that Mep permease involvement in PKA control is different from their role in haploid invasive growth, in which Mep1 sustains and Mep2 inhibits, in a way independent of the ammonium level in the medium.     

Inorganic phosphate is sensed by specific phosphate carriers and acts in concert with glucose as a nutrient signal for activation of the protein kinase A pathway in the yeast Saccharomyces cerevisiae

Yeast cells starved for inorganic phosphate on a glucose-containing medium arrest growth and enter the resting phase G0. We show that re-addition of phosphate rapidly affects well known protein kinase A targets: trehalase activation, trehalose mobilization, loss of heat resistance, repression of STRE-controlled genes and induction of ribosomal protein genes. Phosphate-induced activation of trehalase is independent of protein synthesis and of an increase in ATP. It is dependent on the presence of glucose, which can be detected independently by the G-protein coupled receptor Gpr1 and by the glucose-phosphorylation dependent system. Addition of phosphate does not trigger a cAMP signal. Despite this, lowering of protein kinase A activity by mutations in the TPK genes strongly reduces trehalase activation. Inactivation of phosphate transport by deletion of PHO84 abolishes phosphate signalling at standard concentrations, arguing against the existence of a transport-independent receptor. The non-metabolizable phosphate analogue arsenate also triggered signalling. Constitutive expression of the Pho84, Pho87, Pho89, Pho90 and Pho91 phosphate carriers indicated pronounced differences in their transport and signalling capacities in phosphate-starved cells. Pho90 and Pho91 sustained highest phosphate transport but did not sustain trehalase activation. Pho84 sustained both transport and rapid signalling, whereas Pho87 was poor in transport but positive for signalling. Pho89 displayed very low phosphate transport and was negative for signalling. Although the results confirmed that rapid signalling is independent of growth recovery, long-term mobilization of trehalose was much better correlated with growth recovery than with trehalase activation. These results demonstrate that phosphate acts as a nutrient signal for activation of the protein kinase A pathway in yeast in a glucose-dependent way and they indicate that the Pho84 and Pho87 carriers act as specific phosphate sensors for rapid phosphate signalling.     

Unravelling the activation mechanisms of protein kinase B/Akt

Over the past decade, protein kinase B (PKB, also termed Akt) has emerged as an important signaling mediator between extracellular cues and modulation of gene expression, metabolism, and cell survival. The enzyme is tightly controlled and consequences of its deregulation include loss of growth control and oncogenesis. Recent work has better characterized the mechanism of PKB activation, including upstream regulators and secondary binding partners. This minireview refreshes some old concepts with new twists and highlights current outstanding questions.     

A feedback loop in the polo-like kinase activation pathway

The Xenopus polo-like kinase Plx1 plays important roles during entry into and exit from mitosis (M phase). Previous studies revealed that Plx1 is activated by phosphorylation on serine and threonine residues, and purification of an activating enzyme from mitotic Xenopus egg extracts led to cloning and characterization of Xenopus polo-like kinase kinase (xPlkk1), which can phosphorylate and activate Plx1 in vitro. In the present study, a positive feedback loop between Plx1 and xPlkk1 was shown to result in each kinase phosphorylating and activating the other. Sequencing of radiolabeled xPlkk1 after phosphorylation by Plx1 in vitro identified three phosphorylation sites each spaced three amino acids apart, two of which have the consensus acidic-X-pSer-hydrophobic described for other polo-like kinase substrates. In addition, endogenous xPlkk1 in oocytes was phosphorylated on these sites in M phase but not in interphase. A mutant xPlkk1 in which these three amino acids were changed to alanine (xPlkk1(SA3)) was unable to be phosphorylated or activated in vitro by Plxl. Depletion of Plx1 from oocyte extracts prior to stimulation of the G(2)/M transition blocked the activation of xPlkk1, but depletion of xPlkk1 before stimulation did not block Plx1 activation. These results indicate that xPlkk1 may function downstream as a target of Plx1 rather than as an upstream activating kinase during the G(2)/M transition.     

PDK1 homologs activate the Pkc1-mitogen-activated protein kinase pathway in yeast

PDK1 (phosphoinositide-dependent kinase 1) is a mammalian growth factor-regulated serine/threonine kinase. Using a genetic selection based on a mutant form of the yeast MAP kinase kinase Ste7, we isolated a gene, PKH2, encoding a structurally and functionally conserved yeast homolog of PDK1. Yeast cells lacking both PKH2 and PKH1, encoding another PDK1 homolog, were nonviable, indicating that Pkh1 and Pkh2 share an essential function. A temperature-sensitive mutant, pkh1(D398G) pkh2, was phenotypically similar to mutants defective in the Pkc1-mitogen-activated protein kinase (MAPK) pathway. Genetic epistasis analyses, the phosphorylation of Pkc1 by Pkh2 in vitro, and reduced Pkc1 activity in the pkh1(D398G) pkh2 mutant indicate that Pkh functions upstream of Pkc1. The Pkh2 phosphorylation site in Pkc1 (Thr-983) is part of a conserved PDK1 target motif and essential for Pkc1 function. Thus, the yeast PDK1 homologs activate Pkc1 and the Pkc1-effector MAPK pathway.     

Multiple activation mechanisms of p38alpha mitogen-activated protein kinase

The p38alpha MAPK participates in a variety of biological processes. Activation of p38alpha is mediated by phosphorylation on specific regulatory tyrosine and threonine sites, and the three dual kinases, MAPK kinase 3 (MKK3), MKK4, and MKK6, are known to be the upstream activators of p38alpha. In addition to activation by upstream kinases, p38alpha can autoactivate when interacting with transforming growth factor-beta-activated protein kinase 1-binding protein 1 (TAB1). Here we used MKK3 and MKK6 double knock-out (MKK3/6 DKO) and MKK4/7 DKO mouse embryonic fibroblast (MEF) cells to examine activation mechanisms of p38alpha. We confirmed that the MKK3/6 pathway is a primary mechanism for p38alpha phosphorylation in MEF cells, and we also showed the presence of other p38alpha activation pathways. We show that TAB1-mediated p38alpha phosphorylation in MEF cells did not need MKK3/4/6, and it accounted for a small portion of the total p38alpha phosphorylation that was induced by hyperosmolarity and anisomycin. We observed that a portion of peroxynitrite-induced phospho-p38alpha is associated with an approximately 85-kDa disulfide complex in wild-type MEF cells. Peroxynitrite-induced phosphorylation of p38alpha in the approximately 85-kDa complex is independent from MKK3/6 because only phospho-p38alpha not associated with the disulfide complex was diminished in MKK3/6 DKO cells. In addition, our data suggest interference among different pathways because TAB1 had an inhibitory effect on p38alpha phosphorylation in the peroxynitrite-induced approximately 85-kDa complex. Mutagenesis analysis of the cysteines in p38alpha revealed that no disulfide bond forms between p38alpha and other proteins in the approximately 85-kDa complex, suggesting it is a p38alpha binding partner(s) that forms disulfide bonds, which enable it to bind to p38alpha. Therefore, multiple mechanisms of p38alpha activation exist that can influence each other, be simultaneously activated by a given stimulus, and/or be selectively used by different stimuli in a cell type-specific manner.     

A pathway in the yeast cell division cycle linking protein kinase C (Pkc1) to activation of Cdc28 at START.

In an effort to study further the mechanism of Cdc28 function and cell cycle commitment, we describe here a genetic approach to identify components of pathways downstream of the Cdc28 kinase at START by screening for mutations that decrease the effectiveness of signaling by Cdc28. The first locus to be characterized in detail using this approach was PKC1 which encodes a homolog of the Ca(2+)-dependent isozymes of the mammalian protein kinase C (PKC) superfamily (Levin et al., 1990). By several genetic criteria, we show a functional interaction between CDC28 and PKC1 with PKC1 apparently functioning with respect to bud emergence downstream of START. Consistent with this, activity of the MAP kinase homolog Mpk1 (a putative Pkc1 effector) is stimulated by activation of Cdc28. Furthermore, we demonstrate a cell cycle-dependent hydrolysis of phosphatidylcholine to diacylglycerol (a PKC activator) and choline phosphate at START. Diacylglycerol production is stimulated by Cdc28 in cycling cells and is closely associated with Cdc28 activation at START. These results imply that the activation of Pkc1, which is known to be necessary during bud morphogenesis, is mediated via the CDC28-dependent stimulation of PC-PLC activity in a novel cell cycle-regulated signaling pathway.     

Collagen-induced platelet activation mainly involves the protein kinase C pathway.

This study analyses early biochemical events in collagen-induced platelet activation. An early metabolic event occurring during the lag phase was the activation of PtdIns(4,5)P2-specific phospholipase C. Phosphatidic acid (PtdOH) formation, phosphorylation of P43 and P20, thromboxane B2 (TXB2) synthesis and platelet secretion began after the lag phase, and were similarly time-dependent, except for TXB2 synthesis, which was delayed. Collagen induced extensive P43 phosphorylation, whereas P20 phosphorylation was weak and always lower than with thrombin. The dose-response curves of P43 phosphorylation and granule secretion were similar, and both reached a peak at 7.5 micrograms of collagen/ml, a dose which induced half-maximal PtdOH and TXB2 formation. Sphingosine, assumed to inhibit protein kinase C, inhibited P43 phosphorylation and secretion in parallel. However, sphingosine was not specific for protein kinase C, since a 15 microM concentration, which did not inhibit P43 phosphorylation, blocked TXB2 synthesis by 50%. Sphingosine did not affect PtdOH formation at all, even at 100 microM, suggesting that collagen itself induced this PtdOH formation, independently of TXB2 generation. The absence of external Ca2+ allowed the cleavage of polyphosphoinositides and the accumulation of InsP3 to occur, but impaired P43 phosphorylation, PtdOH and TXB2 formation, and secretion; these were only restored by adding 0.11 microM-Ca2+. In conclusion, stimulation of platelet membrane receptors for collagen initiates a PtdInsP2-specific phospholipase C activation, which is independent of external Ca2+, and might be the immediate receptor-linked response. A Ca2+ influx is indispensable to the triggering of subsequent platelet responses. This stimulation predominantly involves the protein kinase C pathway associated with secretion, and appears not to be mediated by TXB2, at least during its initial stage.     

Novel phosphorylation site markers of protein kinase C delta activation

Protein kinase C delta (PKCdelta) is a Ser/Thr kinase which regulates numerous cellular processes, including proliferation, differentiation, migration and apoptosis. Here, we demonstrate that PKCdelta undergoes in vitro autophosphorylation at three sites within its V3 region (S299, S302, S304), each of which is unique to this PKC isoform and evolutionarily conserved. We demonstrate that S299 and S304 can be phosphorylated in mammalian cells following phorbol ester stimulation and that S299-phosphorylated PKCdelta is localised to both the plasma and nuclear membranes. These data indicate that PKCdelta is phosphorylated upon activation and that phospho-S299 represents a useful marker of the activated enzyme.     

Novel mechanisms of the regulation of protein kinase B in adipocytes; implications for protein kinase A, Epac, phosphodiesterases 3 and 4

Crosstalk between insulin and cAMP signalling pathways has a great impact on adipocyte metabolism. Whilst Protein kinase B (PKB) is a pivotal mediator of insulin action, in some cells regulation of PKB by cAMP has also been demonstrated. Here we provide evidence that, in a phosphatidyl inositol 3-kinase dependent manner, beta3-adrenergic stimulation (using CL316243) in adipocytes induces PKB phosphorylation in the absence of insulin and also potentiates insulin-induced phosphorylation of PKB. Interestingly, insulin- and CL316243-induced PKB phosphorylation was found to be inhibited by pools of cAMP controlled by PDE3B and PDE4 (mainly in the context of insulin), whereas a cAMP pool controlling protein kinase A appeared to mediate stimulation of PKB phosphorylation (mainly in the context of CL316243). Furthermore, an Epac (exchange protein directly activated by cAMP) agonist (8-pCPT-2'-O-Me-cAMP) mimicked the effect of the PDE inhibitors, giving evidence that Epac has an inhibitory effect on PKB phosphorylation in adipocytes. Further, we put the results obtained at the level of PKB in the context of possible downstream signalling components in the regulation of adipocyte metabolism. Thus, we found that overexpression of PKB induced lipogenesis in a PDE3B-dependent manner. Furthermore, overexpression or inhibition of PDE3B was associated with reduced or increased phosphorylation of the key lipogenic enzyme acetyl-CoA carboxylase (ACC), respectively. These PDE3B-dependent effects on ACC correlated with changes in lipogenesis. The Epac agonist, 8-pCPT-2'-O-Me-cAMP, mimicked the effect of PDE3B inhibition on ACC phosphorylation and lipogenesis.