The requirement for the hydrophobic motif phosphorylation of Ypk1 in yeast differs depending on the downstream events, including endocytosis, cell growth, and resistance to a sphingolipid biosynthesis inhibitor, ISP-1

Tanoue, Daisuke; Kobayashi, Takafumi; Sun, Yidi; Fujita, Tetsuro; Takematsu, Hiromu; Kozutsumi, Yasunori

doi:10.1016/j.abb.2005.02.030

Cited by 16 publications

(19 citation statements)

References 43 publications

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“…For example, for yeast Sch9, like mammalian p70 S6K , TORC1 is responsible, whereas for Ypk1 (and Ypk2), like mammalian AKT and SGK, TORC2 is responsible (27,32). It is noteworthy that mutation of the hydrophobic motif in Ypk1 does not diminish its "intrinsic" catalytic activity toward a peptide substrate (16); yet, as we showed here, Ypk1(T662A) is clearly impaired in its activity toward Orm1 and Orm2 both in vitro and in vivo, consistent with the observation that, unlike cells expressing wild-type Ypk1, this mutant is unable to grow when challenged with myriocin (16,17). A similar finding has been made with mammalian AKT.…”

Section: Homeostatic Phosphoregulation Of Orm Proteins By Ypk1 Requiressupporting

confidence: 76%

“…In the Ypk1 paralog, Ypk2, phosphorylation of S641 (in the turn motif), and T659 (in the hydrophobic motif) depends on target of rapamycin protein kinase 2 (Tor2) (11), specifically in the TORC2 complex (27,28), and absence of these modifications prevents optimal ceramide biosynthesis (6). Similarly, we (16) and others (17) found previously that a Ypk1(T662A) mutant grows normally on rich medium, but is unable to grow on the same medium containing myriocin, suggesting that phosphorylation of Ypk1 at T662 in the hydrophobic motif is critical for survival when sphingolipids become limiting. By contrast, the same T662A alteration had no effect on sensitivity of cells to a known TORC1-specific inhibitor, rapamycin (16).…”

Section: Phosphorylation Of Ypk1 By Target Of Rapamycin Protein Kinasmentioning

confidence: 99%

“…In particular, we had previously implicated Ypk1 function in the regulation of sphingolipid biosynthesis in two ways. First, Ypk1 overexpression confers resistance to the SPT inhibitor myriocin, whereas loss of YPK1 causes profound hypersensitivity to this drug (16,17). Second, Ypk1 is a pivotal component of regulatory circuitry that imposes a balance between the pool of complex sphingolipids and the amount of aminophospholipids in the outer leaflet (12).…”

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confidence: 99%

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Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae

Roelants

Breslow

Muir

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

271

424

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The Orm family proteins are conserved integral membrane proteins of the endoplasmic reticulum that are key homeostatic regulators of sphingolipid biosynthesis. Orm proteins bind to and inhibit serine: palmitoyl-coenzyme A transferase, the first enzyme in sphingolipid biosynthesis. In Saccharomyces cerevisiae, Orm1 and Orm2 are inactivated by phosphorylation in response to compromised sphingolipid synthesis (e.g., upon addition of inhibitor myriocin), thereby restoring sphingolipid production. We show here that protein kinase Ypk1, one of an essential pair of protein kinases, is responsible for this regulatory modification. Myriocin-induced hyperphosphorylation of Orm1 and Orm2 does not occur in ypk1Δ cells, and immunopurified Ypk1 phosphorylates Orm1 and Orm2 robustly in vitro exclusively on three residues that are known myriocin-induced sites. Furthermore, the temperature-sensitive growth of ypk1 ts ypk2Δ cells is substantially ameliorated by deletion of ORM genes, confirming that a primary physiological role of Ypk1-mediated phosphorylation is to negatively regulate Orm function. Ypk1 immunoprecipitated from myriocin-treated cells displays a higher specific activity for Orm phosphorylation than Ypk1 from untreated cells. To identify the mechanism underlying Ypk1 activation, we systematically tested several candidate factors and found that the target of rapamycin complex 2 (TORC2) kinase plays a key role. In agreement with prior evidence that a TORC2-dependent site in Ypk1(T662) is necessary for cells to exhibit a wild-type level of myriocin resistance, a Ypk1 (T662A) mutant displays only weak Orm phosphorylation in vivo and only weak activation in vitro in response to sphingolipid depletion. Additionally, sphingolipid depletion increases phosphorylation of Ypk1 at T662. Thus, Ypk1 is both a sensor and effector of sphingolipid level, and reduction in sphingolipids stimulates Ypk1, at least in part, via TORC2-dependent phosphorylation.cell regulation | plasma membrane

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Section: Homeostatic Phosphoregulation Of Orm Proteins By Ypk1 Requiressupporting

confidence: 76%

Section: Phosphorylation Of Ypk1 By Target Of Rapamycin Protein Kinasmentioning

confidence: 99%

mentioning

confidence: 99%

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Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae

Roelants

Breslow

Muir

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

271

424

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“…Thus, in budding yeast, TORC1 specifically recognizes Sch9, the likely ortholog of mammalian S6K1, and TORC2 specifically recognizes Ypk2, a likely ortholog of mammalian Akt and SGK (8,9). Ypk2, as well as the closely related protein Ypk1, has been implicated in regulating several important cellular functions, including receptor-mediated endocytosis, actin polarization, and sphingolipid metabolism, processes that are also linked to TORC2 (9)(10)(11)(12)(13)(14). Given the extreme sequence similarity (∼90%) between Ypk1 and Ypk2, as well as their overlapping functions, it has been assumed that Ypk1 is also a target of TORC2 (9).…”

mentioning

confidence: 99%

Plasma membrane recruitment and activation of the AGC kinase Ypk1 is mediated by target of rapamycin complex 2 (TORC2) and its effector proteins Slm1 and Slm2

Niles

Mogri

Hill

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

133

228

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The yeast AGC kinase orthologs Ypk1 and Ypk2 control several important cellular processes, including actin polarization, endocytosis, and sphingolipid metabolism. Activation of Ypk1/2 requires phosphorylation by kinases localized at the plasma membrane (PM), including the 3-phosphoinositide-dependent kinase 1 orthologs Pkh1/Pkh2 and the target of rapamycin complex 2 (TORC2). Unlike their mammalian counterparts SGK and Akt, Ypk1 and Ypk2 lack an identifiable lipid-targeting motif; therefore, how these proteins are recruited to the PM has remained elusive. To explore Ypk1/2 function, we constructed ATP analog-sensitive alleles of both kinases and monitored global changes in gene expression following their inhibition, where we observed increased expression of stress-responsive target genes controlled by Ca 2+ -dependent phosphatase calcineurin. TORC2 has been shown previously to negatively regulate calcineurin in part by phosphorylating two related proteins, Slm1 and Slm2, which associate with the PM via plextrin homology domains. We therefore investigated the relationship between Slm1 and Ypk1 and discovered that these proteins interact physically and that Slm1 recruits Ypk1 to the PM for phosphorylation by TORC2. We observed further that these steps facilitate subsequent phosphorylation of Ypk1 by Pkh1/2. Remarkably, a requirement for Slm1, can be bypassed by fusing the plextrin homology domain of Slm1 alone onto Ypk1, demonstrating that the essential function of Slm1 is largely attributable to its role in Ypk1 activation. These findings both extend the scope of cellular processes regulated by Ypk1/2 to include negative regulation of calcineurin and broaden the repertoire of mechanisms for membrane recruitment and activation of a protein kinase.

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“…Ypk1, the yeast homolog of the human serum-and glucocorticoid-induced kinase (Sgk1), is a serine/threonine protein kinase known to affect multiple downstream processes, including lipid homeostasis (1)(2)(3)(4)(5), actin dynamics (6, 7), cell wall integrity (6,7), endocytosis (3,8), and lipid flippase activity (1). Ypk1 has a functionally redundant homolog, Ypk2 (Ykr2).…”

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confidence: 99%

Loss of Ypk1, the Yeast Homolog to the Human Serum- and Glucocorticoid-induced Protein Kinase, Accelerates Phospholipase B1-mediated Phosphatidylcholine Deacylation

Surlow

Cooley

Needham

et al. 2014

Journal of Biological Chemistry

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Background: Ypk1 is a protein kinase known to regulate sphingolipid homeostasis. Plb1 deacylates phosphatidylcholine to produce glycerophosphocholine. Results: Loss of Ypk1 or disruption of sphingolipid synthesis by other means elevates Plb1-mediated phosphatidylcholine turnover. Conclusion: Accelerated turnover of phosphatidylcholine compensates for aberrant sphingolipid synthesis. Significance: Sphingolipid synthesis is coordinated with phosphatidylcholine metabolism to maintain membrane lipid homeostasis.

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The requirement for the hydrophobic motif phosphorylation of Ypk1 in yeast differs depending on the downstream events, including endocytosis, cell growth, and resistance to a sphingolipid biosynthesis inhibitor, ISP-1

Cited by 16 publications

References 43 publications

Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae

Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae

Plasma membrane recruitment and activation of the AGC kinase Ypk1 is mediated by target of rapamycin complex 2 (TORC2) and its effector proteins Slm1 and Slm2

Loss of Ypk1, the Yeast Homolog to the Human Serum- and Glucocorticoid-induced Protein Kinase, Accelerates Phospholipase B1-mediated Phosphatidylcholine Deacylation

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