The Tap42-Protein Phosphatase Type 2A Catalytic Subunit Complex Is Required for Cell Cycle-Dependent Distribution of Actin in Yeast

ABSTRACT In Saccharomyces cerevisiae, the Tor proteins mediate a wide spectrum of growth-related cellular processes in response to nutrients. The pleiotropic role of the Tor proteins is mediated, at least in part, by type 2A protein phosphatases (PP2A) and 2A-like protein phosphatases. Tor-mediated signaling activity promotes the interaction of phosphatase-interacting protein Tap42 with PP2A and 2A-like protein phosphatases. The distinct complexes formed between Tap42 and different phosphatases mediate various cellular events and modulate phosphorylation levels of many downstream factors in the Tor pathway in a Tor-dependent and rapamycin-sensitive manner. In this study, we demonstrate that the interaction between Tap42 and the catalytic subunits of PP2A (PP2Ac) is required for cell cycle-dependent distribution of actin. We show that mutations in PP2Ac and Tap42 that perturb the interaction cause random distribution of actin during the cell cycle and that overexpression of the Rho2 GTPase suppresses the actin defects associated with the mutants. Our findings suggest that the Tap42-PP2Ac complex regulates the actin cytoskeleton via a Rho GTPase-dependent mechanism. In addition, we provide evidence that PP2A activity plays a negative role in controlling the actin cytoskeleton and, possibly, in regulation of the G2/M transition of the cell cycle.

[1]  Takeshi Noda,et al.  Tor, a Phosphatidylinositol Kinase Homologue, Controls Autophagy in Yeast* , 1998, The Journal of Biological Chemistry.

[2]  J. Broach,et al.  Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae , 1992, Molecular and cellular biology.

[3]  I. Howald,et al.  TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae. , 1998, Genetics.

[4]  R. Rothstein.  Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. , 1991, Methods in enzymology.

[5]  D. Lew.  Cell-cycle checkpoints that ensure coordination between nuclear and cytoplasmic events in Saccharomyces cerevisiae. , 2000, Current opinion in genetics & development.

[6]  S. Schreiber,et al.  Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Heitman,et al.  Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast , 1991, Science.

[8]  J. Heitman,et al.  TOR Mutations Confer Rapamycin Resistance by Preventing Interaction with FKBP12-Rapamycin (*) , 1995, The Journal of Biological Chemistry.

[9]  B. Haarer,et al.  Immunofluorescence methods for yeast. , 1991, Methods in enzymology.

[10]  Tobias Schmelzle,et al.  TOR, a Central Controller of Cell Growth , 2000, Cell.

[11]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[12]  J. Kunz,et al.  Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression , 1993, Cell.

[13]  Michael N. Hall,et al.  The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors , 1999, Nature.

[14]  X. Zheng,et al.  Tripartite Regulation of Gln3p by TOR, Ure2p, and Phosphatases* , 2000, The Journal of Biological Chemistry.

[15]  M. A. de la Torre-Ruiz,et al.  Regulation of the Cell Integrity Pathway by Rapamycin-sensitive TOR Function in Budding Yeast* , 2002, The Journal of Biological Chemistry.

[16]  C. Berset,et al.  The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast Saccharomyces cerevisiae. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[17]  H. Ronne,et al.  Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis , 1991, Molecular and cellular biology.

[18]  Stuart L. Schreiber,et al.  TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin , 1995, Cell.

[19]  T. Kunkel,et al.  Efficient site-directed mutagenesis using uracil-containing DNA. , 1991, Methods in enzymology.

[20]  M. Mclaughlin,et al.  Interaction between FKBP12-rapamycin and TOR involves a conserved serine residue. , 1994, The Journal of biological chemistry.

[21]  M. Kirschner,et al.  Inhibition of cdc2 activation by INH/PP2A. , 1994, Molecular biology of the cell.

[22]  M. Yanagida,et al.  Distinct, essential roles of type 1 and 2A protein phosphatases in the control of the fission yeast cell division cycle , 1990, Cell.

[23]  J. Heitman,et al.  The TOR signaling cascade regulates gene expression in response to nutrients. , 1999, Genes & development.

[24]  J. V. Gray,et al.  The Protein Kinase C Pathway Is Required for Viability in Quiescence in Saccharomyces cerevisiae , 2002, Current Biology.

[25]  M. Kirschner,et al.  INH, a negative regulator of MPF, is a form of protein phosphatase 2A , 1991, Cell.

[26]  Rodney Rothstein,et al.  Elevated recombination rates in transcriptionally active DNA , 1989, Cell.

[27]  H. Tung,et al.  Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. , 1988, Methods in enzymology.

[28]  M. Stark,et al.  Mutations in the Saccharomyces cerevisiae type 2A protein phosphatase catalytic subunit reveal roles in cell wall integrity, actin cytoskeleton organization and mitosis. , 1997, Genetics.

[29]  Gerald R. Fink,et al.  Guide to yeast genetics and molecular biology , 1993 .

[30]  M. Hall,et al.  TOR2 is required for organization of the actin cytoskeleton in yeast. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[31]  I. Stansfield,et al.  An MBoC Favorite: TOR controls translation initiation and early G1 progression in yeast , 2012, Molecular biology of the cell.

[32]  K. Arndt,et al.  SIT4 protein phosphatase is required for the normal accumulation of SWI4, CLN1, CLN2, and HCS26 RNAs during late G1. , 1992, Genes & development.

[33]  M. Goebl,et al.  CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase , 1991, Molecular and cellular biology.

[34]  M. Ehrmann,et al.  The Saccharomyces cerevisiae gene PPH3 encodes a protein phosphatase with properties different from ppx, PP1 and PP2A , 1994, Yeast.

[35]  K. Arndt,et al.  TIP41 interacts with TAP42 and negatively regulates the TOR signaling pathway. , 2001, Molecular cell.

[36]  K. Arndt,et al.  Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. , 1996, Genes & development.

[37]  T. Powers,et al.  Regulation of ribosome biogenesis by the rapamycin-sensitive TOR-signaling pathway in Saccharomyces cerevisiae. , 1999, Molecular biology of the cell.

[38]  E. Harlow,et al.  Antibodies: A Laboratory Manual , 1988 .

[39]  M. Hall,et al.  Signaling to the actin cytoskeleton. , 1998, Annual review of cell and developmental biology.

[40]  P. Cohen,et al.  Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes. , 1990, The EMBO journal.

[41]  A. Shaw,et al.  The 14-3-3 proteins positively regulate rapamycin-sensitive signaling , 1998, Current Biology.

[42]  K. Arndt,et al.  The SAP, a new family of proteins, associate and function positively with the SIT4 phosphatase , 1996, Molecular and cellular biology.

[43]  Y. Shu,et al.  Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A , 1997, Molecular and cellular biology.

[44]  S. Schreiber,et al.  Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[45]  K. Arndt,et al.  The role of Saccharomyces cerevisiae type 2A phosphatase in the actin cytoskeleton and in entry into mitosis. , 1995, The EMBO journal.

[46]  D. Bergsma,et al.  Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein. , 1991, Molecular and cellular biology.

[47]  Y. Ohya,et al.  The Rho1 effector Pkc1, but not Bni1, mediates signalling from Tor2 to the actin cytoskeleton , 1998, Current Biology.

[48]  J. Broach,et al.  A ceramide-activated protein phosphatase mediates ceramide-induced G1 arrest of Saccharomyces cerevisiae. , 1996, Genes & development.

[49]  M. Mclaughlin,et al.  Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity , 1993, Molecular and cellular biology.

[50]  M. Bickle,et al.  Cell wall integrity modulates RHO1 activity via the exchange factor ROM2 , 1998, The EMBO journal.

[51]  J. Broach,et al.  Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast , 1999, The EMBO journal.

[52]  K. Arndt,et al.  The SIT4 protein phosphatase functions in late G1 for progression into S phase , 1991, Molecular and cellular biology.

[53]  Marc Bickle,et al.  The Yeast Phosphatidylinositol Kinase Homolog TOR2 Activates RHO1 and RHO2 via the Exchange Factor ROM2 , 1997, Cell.