Identification of protein phosphatase 1 in synaptic junctions: dephosphorylation of endogenous calmodulin-dependent kinase II and synapse-enriched phosphoproteins

A calcium/calmodulin-dependent protein kinase termed CaM-kinase II is a major component of synaptic junctions from forebrain and constitutes approximately 12% of total synaptic junction protein. CaM-kinase II phosphorylates at least seven polypeptides that are enriched in synaptic junctions, of which two represent the 50- and 60-kilodalton subunits of the protein kinase. In this report the nature of endogenous protein phosphatases which dephosphorylate each of the seven synaptic junction phosphoproteins was examined. Assays of synaptic junctions and other subcellular fractions from rat forebrain for type-1 and type-2 protein phosphatases revealed that protein phosphatase 1 (PrP-1) was specifically enriched in synaptic junctions with respect to cytosolic fractions. The activity of type-2 protein phosphatases was very low in synaptic junctions. Homogeneous PrP-1 from rabbit skeletal muscle was found to dephosphorylate each of the seven phosphoproteins in synaptic junctions. Inhibitors-1 and -2 were found to inhibit endogenous protein phosphatase activity by 70 to 80%. Since inhibitors-1 and -2 are specific inhibitors of PrP-1, these results indicate that this enzyme accounts for the majority of endogenous protein phosphatase activity in synaptic junctions. Approximately 15% of the protein phosphatase activity in synaptic junctions was type 2A, whereas PrP-2B and PrP-2C accounted for little, if any, of the activity toward endogenous or exogenous phosphoproteins. These results indicate that PrP-1 may be important in controlling the state of phosphorylation of synaptic junction proteins.

[1]  Elmer S. West.  From the U. S. A. , 1965 .

[2]  P. Greengard,et al.  Mammalian brain phosphoproteins as substrates for calcineurin. , 1984, The Journal of biological chemistry.

[3]  P. Greengard,et al.  Depolarizing agents and cyclic nucleotides regulate the phosphorylation of specific neuronal proteins in rat cerebral cortex slices. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Wood,et al.  Immunocytochemical localization of calmodulin and a heat-labile calmodulin-binding protein (CaM-BP80) in basal ganglia of mouse brain , 1980, The Journal of cell biology.

[5]  F. Huang,et al.  Separation and characterization of two phosphorylase phosphatase inhibitors from rabbit skeletal muscle. , 1976, European journal of biochemistry.

[6]  P. Cohen,et al.  The protein phosphatases involved in cellular regulation. 1. Classification and substrate specificities. , 1983, European journal of biochemistry.

[7]  P. Cohen,et al.  The Hormonal Control of Glycogen Metabolism , 1979 .

[8]  L. Glaser,et al.  Resistance to phosphatase of thiophosphorylated epidermal growth factor receptor in A431 membranes. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[9]  T. Yamauchi,et al.  Phosphorylation of microtubule-associated protein 2 by calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues. , 1982, Biochemical and biophysical research communications.

[10]  W. Kerrick,et al.  Chicken gizzard: relation between calcium-activated phosphorylation and contraction. , 1979, Science.

[11]  P. Greengard,et al.  Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes. , 1977, The Journal of biological chemistry.

[12]  C. Klee,et al.  Calcineurin: a calcium- and calmodulin-binding protein of the nervous system. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[13]  P. Kelly,et al.  Synaptic junctions isolated from cerebellum and forebrain: Comparisons of morphological and molecular properties , 1983, Brain Research.

[14]  P. Cohen,et al.  The regulation of glycogen metabolism. Purification and properties of protein phosphatase inhibitor-2 from rabbit skeletal muscle. , 1980, European journal of biochemistry.

[15]  M K Bennett,et al.  Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain. , 1983, The Journal of biological chemistry.

[16]  P. Cohen,et al.  The regulation of glycogen metabolism. Purification and characterisation of protein phosphatase inhibitor-1 from rabbit skeletal muscle. , 1978, European journal of biochemistry.

[17]  D. Hartshorne,et al.  Roles of calcium and phosphorylation in the regulation of the activity of gizzard myosin. , 1978, Biochemistry.

[18]  P. Cohen,et al.  The protein phosphatases involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B) from rabbit skeletal muscle. , 1983, European journal of biochemistry.

[19]  R. Erikson,et al.  Separation of multiple phosphotyrosyl-and phosphoseryl-protein phosphatases from chicken brain. , 1983, The Journal of biological chemistry.

[20]  P. Greengard,et al.  A calcium/calmodulin-dependent protein kinase from mammalian brain that phosphorylates Synapsin I: partial purification and characterization , 1983, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[21]  P. J. Vernon,et al.  Autophosphorylation of Calmodulin‐Kinase II in Synaptic Junctions Modulates Endogenous Kinase Activity , 1984, Journal of neurochemistry.

[22]  P. Kelly,et al.  Cyclic AMP-stimulated protein kinases at brain synaptic junctions. , 1979, The Journal of biological chemistry.

[23]  P. Cohen,et al.  The subunit structure of rabbit-skeletal-muscle phosphorylase kinase, and the molecular basis of its activation reactions. , 1973, European journal of biochemistry.

[24]  H. Schulman.  Phosphorylation of microtubule-associated proteins by a Ca2+/calmodulin- dependent protein kinase , 1984, The Journal of cell biology.

[25]  P. Greengard,et al.  DARPP-32, a dopamine-regulated neuronal phosphoprotein, is a potent inhibitor of protein phosphatase-1 , 1984, Nature.

[26]  P. Cohen,et al.  The protein phosphatases involved in cellular regulation. 4. Classification of two homogeneous myosin light chain phosphatases from smooth muscle as protein phosphatase-2A1 and 2C, and a homogeneous protein phosphatase from reticulocytes active on protein synthesis initiation factor eIF-2 as protein , 1983, European journal of biochemistry.

[27]  P. Siekevitz,et al.  Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity , 1981, The Journal of cell biology.

[28]  T. Yamauchi,et al.  Purification and characterization of the brain calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase. , 1983, European journal of biochemistry.

[29]  L. Witters,et al.  The protein phosphatases involved in cellular regulation. 3. Fatty acid synthesis, cholesterol synthesis and glycolysis/gluconeogenesis. , 1983, European journal of biochemistry.

[30]  P. Kelly,et al.  Evidence that a Cerebellum‐Enriched, Synaptic Junction Glycoprotein Is Related to Fodrin and Resists Extraction with Triton in a Calcium‐Dependent Manner , 1984, Journal of neurochemistry.

[31]  P. Cohen,et al.  Calcineurin is a calcium ion-dependent, calmodulin-stimulated protein phosphatase. , 1983, Biochimica et biophysica acta.

[32]  P. Greengard,et al.  Evidence that the major postsynaptic density protein is a component of a Ca2+/calmodulin-dependent protein kinase. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[33]  E. Krebs,et al.  The isolation and crystallization of rabbit skeletal muscle phosphorylase b. , 1958, The Journal of biological chemistry.

[34]  P. Cohen,et al.  The protein phosphatases involved in cellular regulation. 2. Glycogen metabolism. , 1983, European journal of biochemistry.

[35]  P. Cuatrecasas,et al.  Calmodulin binding to the cytoskeletal neuronal calmodulin-dependent protein kinase is regulated by autophosphorylation. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[36]  P. Kelly,et al.  Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide , 1978, The Journal of cell biology.

[37]  J. Goldenring,et al.  Identification of the Major Postsynaptic Density Protein as Homologous with the Major Calmodulin‐Binding Subunit of a Calmodulin‐Dependent Protein Kinase , 1984, Journal of neurochemistry.

[38]  S. Yang,et al.  Calcineurin is a calmodulin-dependent protein phosphatase. , 1982, Biochemical and biophysical research communications.

[39]  P. Greengard,et al.  Synapsin I (Protein I), a nerve terminal-specific phosphoprotein. II. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes , 1983, The Journal of cell biology.

[40]  P. Greengard,et al.  Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[41]  P. Cohen,et al.  The protein phosphatases involved in cellular regulation. 6. Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles. , 1983, European journal of biochemistry.

[42]  P. Greengard,et al.  Calmodulin-dependent protein kinase and associated substrates in Torpedo electric organ. , 1983, The Journal of biological chemistry.

[43]  P. Cohen,et al.  Separation of two phosphorylase kinase phosphatases from rabbit skeletal muscle. , 1976, European journal of biochemistry.

[44]  P. Kelly,et al.  Subcellular localization of the 52,000 molecular weight major postsynaptic density protein , 1982, Brain Research.

[45]  M K Bennett,et al.  Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[46]  H. Tung,et al.  The catalytic subunits of protein phosphatase-1 and protein phosphatase 2A are distinct gene products. , 1984, European journal of biochemistry.

[47]  P. Cohen,et al.  Protein phosphatases: properties and role in cellular regulation. , 1983, Science.

[48]  P. Kelly,et al.  Calmodulin‐Dependent Protein Phosphorylation in Synaptic Junctions , 1985, Journal of neurochemistry.

[49]  P. Cohen,et al.  The protein phosphatases involved in cellular regulation , 1984 .