Autophosphorylation Sites of the Type II Ca²⁺/Calmodulin-Dependent Protein Kinase: Identification, Regulation of Kinase Activity, and Site-Specific Antibodies

Citation

Patton, Bruce Lowell (1991) Autophosphorylation Sites of the Type II Ca²⁺/Calmodulin-Dependent Protein Kinase: Identification, Regulation of Kinase Activity, and Site-Specific Antibodies. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ete7-xp75. https://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 α and β 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 kinase catalytic activity. In the presence of Ca²⁺ and calmodulin, type II CaM kinase autophosphorylated an homologous residue in the β 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.

Thesis Files