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Autophosphorylation sites on the type II CA2+/calmodulin-dependent protein kinase : identification, regulation of kinase activity, and site-specific antibodies

Citation

Patton, Bruce L. (1991) Autophosphorylation sites on the type II CA2+/calmodulin-dependent protein kinase : identification, regulation of kinase activity, and site-specific antibodies. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-07232007-144526

Abstract

Biochemical and immunological approaches have been developed to study the regulation of the rat neuronal type II Ca2+/calmodulin-dependent protein kinase (type II CaM kinase) by autophosphorylation. This thesis describes the identification of in vitro autophosphorylation sites on the CaM kinase and their role in regulating the catalytic activity of the CaM kinase. In addition, this thesis describes the development of antibodies against the type II CaM kinase that specifically recognize either the autophosphorylated kinase or the nonphosphorylated kinase.

The autophosphorylation sites on in vitro autophosphorylated type II CaM kinase were identified by tryptic phosphopeptide mapping using reverse phase HPLC to isolate individual autophosphorylation sites. The sequence of the purified phosphopeptides was determined by gas phase microsequencing and compared to the known sequences of the kinase subunits, deduced from the cDNAs encoding them. The rates of site-specific autophosphorylation, or dephosphorylation by protein phosphatases, was compared with the rate of change in the Ca2+/ calmodulin-dependence of kinase catalytic activity. In the presence of Ca2+ and calmodulin, type II CaM kinase autophosphorylated an homologous residue in the [alpha] and [beta] subunits of the type II CaM kinase, Thr286 and Thr287, respectively. Phosphorylation of this site correlated with the generation of ca2+ -independent catalytic activity. Removal of free Ca2+ ion from the autophosphorylation reaction resulted in the autophosphorylation of two pairs of homologous residues, Thr305 and Ser314 in the a subunit a and Thr306 and Ser315 in the [beta] subunit. Ser314/315 is resistant to dephosphorylation by purified protein phosphatases 1 and 2A. Selective dephosphorylation of the Thr305/306 autophosphorylation site demonstrated that the presence of phosphate on Thr305/306 inhibits Ca2+/calmodulin-stimulated catalytic activity. The presence of phosphate on Ser314/315 slightly decreases the sensitivity of the kinase to Ca2+/calmodulin.

Antibodies that bind to the type II CaM kinase at the Thr286/287 autophosphorylation site were produced in note and rabbits by immunization with thiophosphorylated and nonphosphorylated peptide haptens. A monoclonal antibody was obtained that specifically recognized the autophosphorylated type IICaM kinase. The monoclonal antibody recognized the Thr286/287 autophosphorylation site. A polyclonal antisera was obtained that, when affinity purified, specifically recognized the nonphosphorylated type II CaM kinase. Autophosphorylation of type II CaM kinase on Thr287 potently inhibited binding of the polyclonal antibodies. The monoclonal antibody and polyclonal antisera recognized type II CaM kinase in immunocytochemical sections and were used to assess the extent and distribution type II CaM kinase autophosphorylation in organotypic cultures of rat brain hippocampal slices. Double immunofluorescence immunocytochemistry with the antibodies specific for phosphorylated and nonphosphorylated type II CaM kinase indicated that most neurons and dendrites contain a mixture of phosphorylated and nonphosphorylated kinase, in varying proportions. Removal of extracellular Ca2+ greatly reduced the immunoreactivity specific for the phosphorylated kinase, implying that the type II CaM kinase phosphorylation state is in dynamic equilibrium in neurons.

Item Type:Thesis (Dissertation (Ph.D.))
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biology
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Kennedy, Mary B.
Thesis Committee:
  • Kennedy, Mary B. (chair)
  • Van Essen, David
  • Lester, Henry A.
  • Tanouye, Mark
  • Patterson, Paul H.
Defense Date:13 May 1991
Record Number:CaltechETD:etd-07232007-144526
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-07232007-144526
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2970
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:01 Aug 2007
Last Modified:26 Dec 2012 02:55

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