VPS34-PI3K Signaling in Saccharomyces cerevisiae
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VPS34-PI3K Signaling in Saccharomyces cerevisiae

PI3Ks (Phosphoinositide-3 Kinases) are heterodimeric lipid kinases that are composed of a regulatory and catalytic subunit that are encoded by different genes (Ref.1). Two distinct VPS34P (Vacuolar Protein-Sorting-34-PI3K (Phosphatidylinositde-3 Kinase)) complexes occur in S. cerevisiae (Saccharomyces cerevisiae) in order to carry out autophagy and CpY (Carboxypeptidase-Y) sorting. VPS34P is activated by VPS15P, a protein kinase. Of the two distinct VPS34P complexes; Complex-I, contains VPS15P, VPS30P/Apg6P (Autophagy Protein-Apg6) and Apg14P, functions in autophagy and the other, Complex-II contains VPS15P, VPS30P/Apg6P (Autophagy Protein-Apg6) and VPS38P which functions in CpY sorting. In general, VPS34P forms at least two multi-subunit PI3K complexes: both contain VPS15P and VPS30P/Apg6P, whereas Apg14P and VPS38P are specific to each. VPS15P-mediated phosphorylation controls binding of VPS34P to VPS38P and Apg14P. PtdIns(3)P (Phosphatidylinositol 3-Phosphate) binding proteins contain a FYVE domain, a subfamily of the cysteine-rich RING motif, which bind directly to PtdIns(3)P. PtdIns(3)P plays an important role in cargo selection at the vesicle budding step. The binding of a cargo protein to the lumenal domain of the receptor transduces a signal through a conformational change that promotes receptor association with and/or activation of the VPS15P. Activation of VPS15P leads to activation of the VPS34P . VPS34P -mediated PtdIns(3)P production recruits effector proteins that function in budding (Ref.2 & 3).

VPS34P is phosphorylated predominately on serine in vivo. VPS34P utilizes PtdIns (Phosphatidylinositol) but not PIP (Phosphatidylinositol 4-Phosphate) or PIP2 (Phosphatidylinositol 4,5-bisphosphate). VPS15P and VPS34P act together within a membrane-associated complex to facilitate the delivery of proteins to the vacuole. VPS34P is able to catalyze the transfer of phosphate from ATP to both lipid (PtdIns) and protein (itself) substrates. VPS34P has strict substrate specificity for PtdIns. Formation of PtdIns(3)P through the action of PtdIns and VPS34P function to regulate intracellular protein trafficking in eukaryotic cells. Thus in yeast, the only detectable PI3K is VPS34P , which catalyses the conversion of PtdIns to PtdIns(3)P (Ref.4,5 & 6). The yeast Sch9, a serine/threonine protein kinase and an Akt (v-Akt Murine Thymoma Viral Oncogene Homolog)/PKB (Protein Kinase-B) homolog, activated by cAMP (Cyclic Adenosine 5-Monophosphate), regulates longevity and stress resistance in yeast. Mammalian Akt/PKB contains a PH (Pleckstrin Homology) domain which is able to bind PIP3 (Phosphatidylinositol 3,4,5-trisphosphate) and PtdIns(3,4)P2 (Phosphatidylinositol 3,4-bisphosphate). Sch9, on the other hand, does not have a PH-domain but contains a C2-domain. C2-domains are known to bind phospholipids. Sch9, as well as its C2-domain, interact with vesicles containing phosphatidylcholine/ phosphatidylserine. The COOH-terminal region of Sch9 is highly homologous to the AGC family of serine/threonine kinases, which includes Akt/PKB, whereas the NH2-terminal region contains a C2-phospholipid and calcium binding motif (Ref.7). The VPS34P -VPS15P kinase complex is an important regulator of protein sorting in eukaryotic cells. The C-terminus of VPS34P is both necessary and sufficient for the interaction with VPS15P. The VPS34P -type kinases constitute the Class-III family of PI3K. The VPS34P motif, encompassing residues 857-864, is unique to this class of PI3K. Only the Class-III PI3Ks interact with a VPS15P-like protein kinase. The other PI3K either interact with a different set of adaptor proteins or work alone. PI3Ks phosphorylate PtdIns and more highly phosphorylated derivatives of this phospholipid at the 3 position of the inositol ring (Ref.8). Glucose and other nutrients activate Sch9 via the GPR1 (G-Protein-Coupled Receptor GPR1) coupled to Gpa2 (G-Protein-Alpha Subunit). The Growth Factor Receptor signaling induced by Growth Factors is negatively regulated by TEP1 (Tensin-Like Phosphatase-1) in S. cerevisiae. TEP1 inhibits the downstream functions mediated by the VPS34P pathway, such as activation of Sch9, cell survival and cell proliferation. TEP1 plays a role in the trafficking of dityrosine to the outside of the spore wall during the diploid-specific process of sporulation (Ref.9, 10 & 11). 

The TOR (Target of Rapamycin) kinases, TOR1 and TOR2, also regulate responses to nutrients, including sporulation, autophagy, mating, and ribosome biogenesis. TOR inhibition by rapamycin induces expression of nitrogen source utilization genes controlled by the Ure2 repressor and the transcriptional regulator for positive nitrogen regulation Gln3, and globally represses ribosomal protein expression. Ure2 is a phosphoprotein in vivo that is rapidly dephosphorylated in response to rapamycin or nitrogen limitation. The TOR cascade plays a prominent role in regulating transcription in response to nutrients in addition to its known roles in regulating translation, ribosome biogenesis and amino acid permease stability. The yeast Tap42 (Two A-Phosphatase Associated Protein-42KD) protein is implicated as target of the TOR pathway. Tap42 is an essential protein that associates with PP2A (Protein Phosphatase-2A) subunits and regulates translation initiation in yeast. TOR also regulates the NPR1, a serine/threonine protein kinase that is required for the stabilization or degradation of amino acid permeases in response to nutrients. Rapamycin represses transcription of rRNA (Ribosomal RNA) and tRNA (Transfer RNA) by inhibiting RNA Pol (RNA Polymerases)-I and III and inhibits ribosome biogenesis. Signaling cascades enable yeast cells to shift from the utilization of abundant levels of good nitrogen sources to either poorer nitrogen sources or limiting concentrations of good nitrogen sources. Glutamine synthetase plays a central role in nitrogen metabolism by converting ammonium to glutamine. Intracellular glutamine is sensed by a pathway involving the Ure2 repressor, which binds and inhibits glutamine synthetase (Gln1) and the transcriptional activator, Gln3. During poor or limiting nitrogen sources, intracellular glutamine decreases, Ure2 is inactivated, and glutamine synthetase and Gln3 are activated, increasing glutamine and inducing nitrogen utilization genes. Gln3 activates genes encoding glutamine synthetase (Gln1), glutamate synthase (Glt1), glutamate dehydrogenases (GDh1 (Glutamate Dehydrogenase-NADP) and GDh2 (Glutamate Dehydrogenase-NAD)), permeases for nitrogenous compounds GAP1 (General Amino acid Permease), a general amino acid permease and Mep2, a high affinity ammonium permease; enzymes involved in nitrogen source metabolism and transcription factors that regulate gene expression. The Ure2, Gln3, NPR1 and Npi1/Rsp5 nitrogen source regulators alter sensitivity to rapamycin by regulating TOR1 or TOR2 expression, or as regulators or effectors of the TOR cascade (Ref.12 & 13). In particular, a functional TOR pathway in S. cerevisiae is required for continued transcription of r-protein (Ribosomal Protein) genes, as well as for the synthesis and processing of 35S precursor rRNA. This pathway is essential for modulation of r-protein gene expression in response to changes in nutrient conditions. Thus in yeast, TOR signaling couples nutrient availability to the transcription of genes involved in the formation of ribosomes. The TOR pathway is directly involved in the regulation of RNA Pol-I, Pol-II and Pol-III activity. These polymerases are essential for the synthesis of 35S rRNA (Pol-I) and 5S rRNA as well as tRNA (Pol-III) (Ref.14). 

In S. cerevisiae the PKC (Protein Kinase-C)-mediated MAPK (Mitogen-Activated Protein Kinase) pathway is regulated by TOR function because upon specific TOR1 and TOR2 inhibition by rapamycin, Mpk1/SLT2 is activated rapidly in a process mediated by SIT4 and Tap42. Osmotic stabilization of the plasma membrane prevents Mpk1 activation by rapamycin. This process involves activation of cell surface sensors (Wsc1/Slg1 and Mid2), Rom2 and downstream elements of the Mitogen-Activated Protein Kinase cascade. In S. cerevisiae cells, the TOR proteins promote association between the SIT4 and Tap42 proteins under favorable nutrient conditions. Rapamycin also induces depolarization of the actin cytoskeleton through the TOR proteins, SIT4 and Tap42, in an osmotically suppressible manner. TOR2 plays an additional essential function that is not shared by TOR1. The TOR2 essential function has been related to the organization of the actin cytoskeleton. The PKC pathway maintains cell integrity by monitoring the cell wall state. It is accepted that the PKC pathway senses cell wall damage and plasma membrane stress through Mid2 and the Wsc family of cell surface sensors, which directly transmit the signal to Rom2 and Rho1. Among other targets, Rho1 directly up-regulates the glucan synthase machinery and is also needed to stimulate PKC1 (Protein Kinase-C1) activity allosterically. In turn, PKC1 activates a module of MAPKKK (MAPK Kinase Kinase) Bck1, the redundant MAPKKs (MAPK Kinases) Mkk1, Mkk2 and Mpk1/SLT2. Mpk1 is phosphorylated on both Thr190 and Tyr192 residues, thus causing a conformational switch that result in its activation. All of the putative phosphorylation sites are conserved in yeast PKC1 and phosphorylation by phosphoinositide-dependent kinases, along with activation by Rho1, plays an important role in regulating PKC1 activity. However, a crucial difference arises in yeast: contrary to what occur in mammalian cells, the TOR shared function negatively affects PKC1 activity. Thus, yeast and mammals have evolved different mechanisms for TOR control over PKC activity (Ref.15).