Appropriate regulation of neuronal function, such as synaptic plasticity, depends in part on neurotransmitter receptors and ion channels, especially those localized in synapses. Phosphorylation/ dephosphorylation of serines and/or threonines in the receptor/ion channel subunits is a major mechanism for their regulation; precise modulation of protein kinase and protein phosphatase activities is therefore essential for learning and memory. For example, activation of type 1 protein phosphatase catalytic subunit (PP1C) isoforms localized in dendritic spine sis likely important in the induction of long-term depression. We characterized four rat brain cytoskeletal, isoform- selective, PP1C-binding proteins (PP1bps) and have purified an actin- associated PP1 holoenzyme (PP1A) containing PP1bp134, PP1bp175 and PP1C. We hypothesize that association of PP1C with these PP1bps specifically targets and regulates neuronal PP1 activity. Three complementary Specific Aims are proposed, which will identify PP1bp134 and PP1bp175 and begin to characterize their physiological role: 1. Characterization and cloning of PP1bps. The cDNAs encoding PP1bps present in PP1A (PP1bp134, PP1bp175) will be isolated. The authenticity of cDNAs will be verified by expression of PP1bp activity in heterologous systems, and by immunological identification of the cloned proteins in PP1A. Expression patterns of PP1C isoform and novel PP1bp mRNAs in tissues, brain regions and during development will be compared. 2. Characterization of the interaction of PP1C, with PP1bps. A. Domain interactions in vitro: Deletion, truncation, and site-directed PP1bp mutants (expressed in bacteria) will be used to identify the minimal isoform selective PP1C binding domain using multiple binding assays. B. Interactions in vivo: Interaction of PP1bps with PP1C isoforms in brain region extracts, brain slices and cultured neurons will be elucidated by co-immunoprecipitation, immunoblotting subcellular fractions, and immunofluorescent confocal microscopy. Effects of wild-type and mutated PP1bps on PP1C localization in undifferentiated heterologous cells (e.g., HEK293, NG108, PC12) and during neuronal differentiation, and on neuronal differentiation itself, will be investigated. 3. Regulation of PP1A. The regulation of PP1bp binding to PP1C and actin by Ca2+/calmodulin-dependent protein kinase II will be investigated and specific phosphorylation sites will be identified. Phosphorylation of PP1bps will be examine din intact cells/neurons and the effects on PP1C/PP1bp localization will be elucidated.
These Aims provide novel insights into the physiological regulation of PP1, a key modulator of synaptic function.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS037508-03
Application #
6330545
Study Section
Special Emphasis Panel (ZRG1-MDCN-1 (01))
Program Officer
Michel, Mary E
Project Start
1998-12-03
Project End
2002-11-30
Budget Start
2000-12-01
Budget End
2001-11-30
Support Year
3
Fiscal Year
2001
Total Cost
$196,934
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Baucum 2nd, Anthony J; Strack, Stefan; Colbran, Roger J (2012) Age-dependent targeting of protein phosphatase 1 to Ca2+/calmodulin-dependent protein kinase II by spinophilin in mouse striatum. PLoS One 7:e31554
Brown, Abigail M; Deutch, Ariel Y; Colbran, Roger J (2005) Dopamine depletion alters phosphorylation of striatal proteins in a model of Parkinsonism. Eur J Neurosci 22:247-56
Bordelon, Jill R; Smith, Yoland; Nairn, Angus C et al. (2005) Differential localization of protein phosphatase-1alpha, beta and gamma1 isoforms in primate prefrontal cortex. Cereb Cortex 15:1928-37
Colbran, Roger J (2004) Targeting of calcium/calmodulin-dependent protein kinase II. Biochem J 378:1-16
Colbran, Roger J; Brown, Abigail M (2004) Calcium/calmodulin-dependent protein kinase II and synaptic plasticity. Curr Opin Neurobiol 14:318-27
Carmody, Leigh C; Bauman, Patricia A; Bass, Martha A et al. (2004) A protein phosphatase-1gamma1 isoform selectivity determinant in dendritic spine-associated neurabin. J Biol Chem 279:21714-23
Colbran, Roger J (2004) Protein phosphatases and calcium/calmodulin-dependent protein kinase II-dependent synaptic plasticity. J Neurosci 24:8404-9
Colbran, Roger J; Carmody, Leigh C; Bauman, Patricia A et al. (2003) Analysis of specific interactions of native protein phosphatase 1 isoforms with targeting subunits. Methods Enzymol 366:156-75
Kloeker, Susanne; Reed, Robin; McConnell, Jamie L et al. (2003) Parallel purification of three catalytic subunits of the protein serine/threonine phosphatase 2A family (PP2A(C), PP4(C), and PP6(C)) and analysis of the interaction of PP2A(C) with alpha4 protein. Protein Expr Purif 31:19-33
Brady, Ashley E; Wang, Qin; Colbran, Roger J et al. (2003) Spinophilin stabilizes cell surface expression of alpha 2B-adrenergic receptors. J Biol Chem 278:32405-12

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