Cellular Mechanisms Regulating Protein Phosphatase-1

Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosphatases (PP1). To define the molecular basis for PP1 inhibition by I-1 and I-2 charged-to-alanine substitutions in the Saccharomyces cerevisiae, PP1 catalytic subunit (GLC7), were analyzed. Two PP1 mutants, E53A/E55A and K165A/E166A/K167A, showed reduced sensitivity to I-2 when compared with wild-type PP1. Both mutants were effectively inhibited by I-1. Two-hybrid analysis and coprecipitation or pull-down assays established that wild-type and mutant PP1 catalytic subunits bound I-2 in an identical manner and suggested a role for the mutated amino acids in enzyme inhibition. Inhibition of wild-type and mutant PP1 enzymes by full-length I-2(1–204), I-2(1–114), and I-2(36–204) indicated that the mutant enzymes were impaired in their interaction with the N-terminal 35 amino acids of I-2. Site-directed mutagenesis of amino acids near the N terminus of I-2 and competition for PP1 binding by a synthetic peptide encompassing an I-2 N-terminal sequence suggested that a PP1 domain composed of amino acids Glu-53, Glu-55, Asp-165, Glu-166, and Lys-167 interacts with the N terminus of I-2. This defined a novel regulatory interaction between I-2 and PP1 that determines I-2 potency and perhaps selectivity as a PP1 inhibitor.

[1]  P. Cohen,et al.  Drosophila melanogaster protein phosphatase inhibitor‐2: identification of a site important for PP1 inhibition , 1999, FEBS letters.

[2]  L. Alphey,et al.  The chaperone-like properties of mammalian inhibitor-2 are conserved in a Drosophila homologue. , 1999, Biochemistry.

[3]  S. Shenolikar,et al.  Importance of the β12-β13 Loop in Protein Phosphatase-1 Catalytic Subunit for Inhibition by Toxins and Mammalian Protein Inhibitors* , 1999, The Journal of Biological Chemistry.

[4]  S. Shenolikar,et al.  Molecular Determinants of Nuclear Protein Phosphatase-1 Regulation by NIPP-1* , 1999, The Journal of Biological Chemistry.

[5]  T Watanabe,et al.  Characterization of the Inhibition of Protein Phosphatase-1 by DARPP-32 and Inhibitor-2* , 1999, The Journal of Biological Chemistry.

[6]  P. Cohen,et al.  Characterisation of a novel Drosophila melanogaster testis specific PP1 inhibitor related to mammalian inhibitor‐2: identification of the site of interaction with PP1 , 1998, FEBS letters.

[7]  S. Zhao,et al.  Identification and characterization of the human HCG V gene product as a novel inhibitor of protein phosphatase-1. , 1998, Biochemistry.

[8]  S. Shenolikar,et al.  Inhibitor-1 Interaction Domain That Mediates the Inhibition of Protein Phosphatase-1* , 1998, The Journal of Biological Chemistry.

[9]  S. Shenolikar,et al.  Gating of CaMKII by cAMP-regulated protein phosphatase activity during LTP. , 1998, Science.

[10]  S. Shenolikar,et al.  Inhibitor-1, a regulator of protein phosphatase 1 function. , 1998, Methods in molecular biology.

[11]  Y. Kakinoki,et al.  Multisite Phosphorylation and the Nuclear Localization of Phosphatase Inhibitor 2-Green Fluorescent Protein Fusion Protein during S Phase of the Cell Growth Cycle* , 1997, The Journal of Biological Chemistry.

[12]  E. Y. Lee,et al.  A Protein Phosphatase-1-binding Motif Identified by the Panning of a Random Peptide Display Library* , 1997, The Journal of Biological Chemistry.

[13]  M. Eto,et al.  Molecular cloning of a novel phosphorylation‐dependent inhibitory protein of protein phosphatase‐1 (CPI17) in smooth muscle: its specific localization in smooth muscle 1 , 1997 .

[14]  Philip R. Cohen,et al.  Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1 , 1997, The EMBO journal.

[15]  P. Greengard,et al.  Characterization of the interaction between DARPP-32 and protein phosphatase 1 (PP-1): DARPP-32 peptides antagonize the interaction of PP-1 with binding proteins. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[16]  A. Bloecher,et al.  Alanine-scanning mutagenesis of protein phosphatase type 1 in the yeast Saccharomyces cerevisiae. , 1997, Genetics.

[17]  T. Sugimura,et al.  Identification and characterization of three isotypes of protein phosphatase inhibitor-2 and their expression profiles during testis maturation in rats. , 1996, European journal of biochemistry.

[18]  Philip R. Cohen,et al.  Further evidence that inhibitor‐2 acts like a chaperone to fold PP1 into its native conformation , 1996, FEBS letters.

[19]  S. Endo,et al.  Multiple structural elements define the specificity of recombinant human inhibitor-1 as a protein phosphatase-1 inhibitor. , 1996, Biochemistry.

[20]  S. Elledge,et al.  Protein phosphatase 1 interacts with p53BP2, a protein which binds to the tumour suppressor p53 , 1995, FEBS letters.

[21]  M. Bollen,et al.  Molecular Cloning of NIPP-1, a Nuclear Inhibitor of Protein Phosphatase-1, Reveals Homology with Polypeptides Involved in RNA Processing (*) , 1995, The Journal of Biological Chemistry.

[22]  H. Tung,et al.  Regulation of chromosome segregation by Glc8p, a structural homolog of mammalian inhibitor 2 that functions as both an activator and an inhibitor of yeast protein phosphatase 1 , 1995, Molecular and cellular biology.

[23]  B. Hemmings,et al.  Serine/threonine protein phosphatases. , 1995, The Biochemical journal.

[24]  Paul Greengard,et al.  Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1 , 1995, Nature.

[25]  J. Byrne,et al.  Protein Phosphatase‐1 Regulates Outward K+ Currents in Sensory Neurons of Aplysia californica , 1995, Journal of neurochemistry.

[26]  A. Depaoli-Roach,et al.  Domains of phosphatase inhibitor-2 involved in the control of the ATP-Mg-dependent protein phosphatase. , 1994, The Journal of biological chemistry.

[27]  J R Feramisco,et al.  Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3T3 fibroblasts , 1994, Molecular and cellular biology.

[28]  R. Malenka,et al.  Involvement of a calcineurin/ inhibitor-1 phosphatase cascade in hippocampal long-term depression , 1994, Nature.

[29]  J. Pringle,et al.  Characterization of glycogen-deficient glc mutants of Saccharomyces cerevisiae. , 1994, Genetics.

[30]  A. Depaoli-Roach,et al.  Molecular mechanism of the synergistic phosphorylation of phosphatase inhibitor-2. Cloning, expression, and site-directed mutagenesis of inhibitor-2. , 1994, The Journal of biological chemistry.

[31]  P. Cohen,et al.  Inhibitor-2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase-1 into a conformation with the specificity and regulatory properties of the native enzyme. , 1993, European journal of biochemistry.

[32]  S. Elledge,et al.  The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. , 1993, Genes & development.

[33]  M. Hagiwara,et al.  Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB , 1992, Cell.

[34]  A. Depaoli-Roach,et al.  Fluorescence studies on the interaction of inhibitor 2 and okadaic acid with the catalytic subunit of type 1 phosphoprotein phosphatases. , 1991, Biochemistry.

[35]  J. Labbé,et al.  Cell cycle oscillation of phosphatase inhibitor-2 in rat fibroblasts coincident with p34 cdc2 restriction , 1990, Nature.

[36]  P. Cohen,et al.  Remarkable similarities between yeast and mammalian protein phosphatases , 1989, FEBS letters.

[37]  V. Dombrádi,et al.  Purification of the catalytic subunit of protein phosphatase-1 from Drosophila melanogaster. , 1987, Biochemical and biophysical research communications.

[38]  H. Tung,et al.  Protein phosphorylation and oocyte maturation. II. Inhibition of starfish oocyte maturation by intracellular microinjection of protein phosphatases 1 and 2A and alkaline phosphatase. , 1986, Experimental cell research.

[39]  J. Maller,et al.  In vivo actions of protein phosphatase inhibitor‐2 in Xenopus oocytes , 1982, FEBS letters.

[40]  E. Fischer,et al.  Phosphorylase phosphatase complex from skeletal muscle. Activation of one of two catalytic subunits by manganese ions. , 1980, Biochemistry.

[41]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .