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
Tóm tắt
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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Tài liệu tham khảo
S Wullschleger, R Loewith, MN Hall, TOR signaling in growth and metabolism. Cell 124, 471–484 (2006).
JR Rohde, R Bastidas, R Puria, ME Cardenas, Nutritional control via Tor signaling in Saccharomyces cerevisiae. Curr Opin Microbiol 11, 153–160 (2008).
R Loewith, et al., Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell 10, 457–468 (2002).
KP Wedaman, et al., Tor kinases are in distinct membrane-associated protein complexes in Saccharomyces cerevisiae. Mol Biol Cell 14, 1204–1220 (2003).
A Reinke, et al., TOR complex 1 includes a novel component, Tco89p (YPL180w), and cooperates with Ssd1p to maintain cellular integrity in Saccharomyces cerevisiae. J Biol Chem 279, 14752–14762 (2004).
DD Sarbassov, DA Guertin, SM Ali, DM Sabatini, Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098–1101 (2005).
K Hara, et al., Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110, 177–189 (2002).
J Urban, et al., Sch9 is a major target of TORC1 in Saccharomyces cerevisiae. Mol Cell 26, 663–674 (2007).
Y Kamada, et al., Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization. Mol Cell Biol 25, 7239–7248 (2005).
FM Roelants, PD Torrance, N Bezman, J Thorner, Pkh1 and Pkh2 differentially phosphorylate and activate Ypk1 and Ykr2 and define protein kinase modules required for maintenance of cell wall integrity. Mol Biol Cell 13, 3005–3028 (2002).
AK deHart, JD Schnell, DA Allen, L Hicke, The conserved Pkh-Ypk kinase cascade is required for endocytosis in yeast. J Cell Biol 156, 241–248 (2002).
D Tanoue, et al., 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. Arch Biochem Biophys 437, 29–41 (2005).
S Aronova, et al., Regulation of ceramide biosynthesis by TOR complex 2. Cell Metab 7, 148–158 (2008).
T Schmelzle, SB Helliwell, MN Hall, Yeast protein kinases and the RHO1 exchange factor TUS1 are novel components of the cell integrity pathway in yeast. Mol Cell Biol 22, 1329–1339 (2002).
D Berchtold, TC Walther, TORC2 plasma membrane localization is essential for cell viability and restricted to a distinct domain. Mol Biol Cell 20, 1565–1575 (2009).
MP Scheid, PA Marignani, JR Woodgett, Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B. Mol Cell Biol 22, 6247–6260 (2002).
AC Pao, et al., NH2 terminus of serum and glucocorticoid-regulated kinase 1 binds to phosphoinositides and is essential for isoform-specific physiological functions. Am J Physiol Renal Physiol 292, F1741–F1750 (2007).
A Audhya, et al., Genome-wide lethality screen identifies new PI4,5P2 effectors that regulate the actin cytoskeleton. EMBO J 23, 3747–3757 (2004).
M Fadri, A Daquinag, S Wang, T Xue, J Kunz, The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2. Mol Biol Cell 16, 1883–1900 (2005).
JM Mulet, DE Martin, R Loewith, MN Hall, Mutual antagonism of target of rapamycin and calcineurin signaling. J Biol Chem 281, 33000–33007 (2006).
M Tabuchi, A Audhya, AB Parsons, C Boone, SD Emr, The phosphatidylinositol 4,5-biphosphate and TORC2 binding proteins Slm1 and Slm2 function in sphingolipid regulation. Mol Cell Biol 26, 5861–5875 (2006).
AM Stathopoulos, MS Cyert, Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast. Genes Dev 11, 3432–3444 (1997).
RM Biondi, A Kieloch, RA Currie, M Deak, DR Alessi, The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB. EMBO J 20, 4380–4390 (2001).
Q Yang, K Inoki, T Ikenoue, KL Guan, Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity. Genes Dev 20, 2820–2832 (2006).
Y Liu, K Shah, F Yang, L Witucki, KM Shokat, Engineering Src family protein kinases with unnatural nucleotide specificity. Chem Biol 5, 91–101 (1998).
K Shokat, M Velleca, Novel chemical genetic approaches to the discovery of signal transduction inhibitors. Drug Discov Today 7, 872–879 (2002).
DP Matheos, TJ Kingsbury, US Ahsan, KW Cunningham, Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae. Genes Dev 11, 3445–3458 (1997).
FM Roelants, AG Baltz, AE Trott, S Fereres, J Thorner, A protein kinase network regulates the function of aminophospholipid flippases. Proc Natl Acad Sci USA 107, 34–39 (2010).
A Schmidt, T Schmelzle, MN Hall, The RHO1-GAPs SAC7, BEM2 and BAG7 control distinct RHO1 functions in Saccharomyces cerevisiae. Mol Microbiol 45, 1433–1441 (2002).
Y Sun, et al., Sli2 (Ypk1), a homologue of mammalian protein kinase SGK, is a downstream kinase in the sphingolipid-mediated signaling pathway of yeast. Mol Cell Biol 20, 4411–4419 (2000).
C Kamble, S Jain, E Murphy, K Kim, Requirements of Slm proteins for proper eisosome organization, endocytic trafficking and recycling in the yeast Saccharomyces cerevisiae. J Biosci 36, 79–96 (2011).
H Yu, et al., High-quality binary protein interaction map of the yeast interactome network. Science 322, 104–110 (2008).
NJ Krogan, et al., Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440, 637–643 (2006).
YG Ni, et al., FoxO transcription factors activate Akt and attenuate insulin signaling in heart by inhibiting protein phosphatases. Proc Natl Acad Sci USA 104, 20517–20522 (2007).
RD Geitz, RA Woods, Transformation of yeast by the lithium acetate/single-stranded carrier DNA/PEG method. Methods in Microbiology, eds AJP Brown, MF Tuite (Academic Press, New York) Vol 26 (1998).
I Dilova, S Aronova, JC Chen, T Powers, Tor signaling and nutrient-based signals converge on Mks1p phosphorylation to regulate expression of Rtg1.Rtg3p-dependent target genes. J Biol Chem 279, 46527–46535 (2004).