Abstract
CaM kinase-Gr is a Ca2+/calmodulin-dependent protein kinase that is enriched in brain and thymus. The enzyme was isolated from rat cerebellum, which contained alpha (M(r) 65,000) and beta (M(r) 67,000) polypeptides, and rat forebrain, which contained only the alpha polypeptide. Both enzyme preparations readily underwent autophosphorylation with dramatic up-regulation of their Ca2+/calmodulin-dependent, as well as-independent, activity. Autophosphorylation also caused a characteristic retardation in the electrophoretic gel mobility of the alpha and beta polypeptides. Treatment of autophosphorylated CaM kinase-Gr with acid phosphatase fully dephosphorylated the enzyme and reversed the changes in electrophoretic migration of both polypeptides. Phosphopeptide mapping indicated that the alpha and beta polypeptides were phosphorylated on identical or homologous sites, which probably induces similar structural and catalytic modifications in the two polypeptides. The actual site(s) of autophosphorylation was determined by the purification and amino acid sequencing of tryptic peptides from 32P-labeled CaM kinase-Gr. The major site of autophosphorylation was localized to a novel N-terminal domain, which is rich in Ser/Thr/Pro residues. The functional and structural studies on CaM kinase-Gr autophosphorylation imply that the enzyme is comprised of two regulatory domains, one on either side of a catalytic domain, followed by a C-terminal, putative association domain. The properties of such a structural model are discussed.
Highlights
CaM kinase-Gr phosphorylates several polypeptides and peptide substrates, which are phosphorylated by CaM kinase-I1 or cyclic AMP-dependentprotein kinase [1, 7], implying a measure of promiscuity in its catalytic selectivity
The enzyme is usually expressed as a M, 65,000 01 polypeptide in forebrain, thymus, spleen, and testis, whereas mature cerebellar granule cells express both the a polypeptide and a M, 67,000 /3 polypeptide [1, 4]
Reduction-alkylation was performed by the addition of 5 pl of 45 mM DTT, heating at 50 "C for30 min, followed by the addition of 5 p1 of 0.1 M iodoacetabeen investigated, it did appear that 50% or more of each component polypeptide underwent a gel-mobility shift
Summary
Buffer B; 95-110 min, 75-98% buffer B. The forebrain CaM kinase-Gr preparation did contain a small amountof an immunoreactive componenwt hich migrates slightly above the a-polypeptide Sional phosphopeptide mapping of 32P-labeled a and p polypeptides (Fig. 3) insofar as trypsin digestion led to the generation of similar setsof major and minor32P-labeledpeptides from the a and p polypeptides. Identified by amino acid sequencing and/or FAB-MS are designated TI-T16 in panel A. rence of similar autophosphorylation sites on the a and /3 polypeptides prompted us to investigate the actual site(s)of phosphate incorporation in greaterdetail. When the67.3-min fraction was analyzed by FABMS andby amino acid sequencing, it proved to be a mixture phosphatase (panel A ). Panel C is similar to panel B to resolve the radioactive peptide(s) from the non-radioactive except that the enzyme polypeptides were visualized by immunoblot- components, a portion of the 67.3-min peak was rechromating. 1 no PAPDAPLKIA I ' DFGLSKIVEH QVWKTVCGT PGYCAPEILR GCAYGPEVDM WSVGIITYIL LCGFEPFYDE
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