Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues

S. Shenolikar, R. Lickteig, D. G. Hardie, Thomas Soderling, R. M. Hanley, P. T. Kelly

Research output: Contribution to journalArticle

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Abstract

Calcium/calmodulin-dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross-reactivity with affinity-purified polyclonal antibodies to the 50-kDa catalytic subunit of the brain calmodulin-dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One-dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases with S. aureus V8 protease, and two-dimensional fingerprints of the 125I-labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56-kDa and/or 58-kDa polypeptides of the skeletal muscle calmodulin-dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50-kDa and 60-kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two-dimensional peptide fingerprints, indicated considerable structural homology among the 50-kDa and 60-kDa subunits of the brain calmodulin-dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51-kDa subunit, skeletal muscle 56-kDa, and the brain 50-kDa and 60-kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin-dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3-5 mol phosphate/mol protein. The two-dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of 32P-labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin-dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin-dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin-dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.

Original languageEnglish (US)
Pages (from-to)739-747
Number of pages9
JournalEuropean Journal of Biochemistry
Volume161
Issue number3
DOIs
StatePublished - 1986
Externally publishedYes

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Calcium-Calmodulin-Dependent Protein Kinases
Calmodulin
Protein Kinases
Isoenzymes
Liver
Brain
Tissue
Muscle
Peptides
Phosphopeptides
Skeletal Muscle
Synapsins
Catalytic Domain
Proteins
Enzymes
Digestion
Proteolysis
Rabbits
Muscle Proteins
Molecular mass

ASJC Scopus subject areas

  • Biochemistry

Cite this

Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues. / Shenolikar, S.; Lickteig, R.; Hardie, D. G.; Soderling, Thomas; Hanley, R. M.; Kelly, P. T.

In: European Journal of Biochemistry, Vol. 161, No. 3, 1986, p. 739-747.

Research output: Contribution to journalArticle

Shenolikar, S. ; Lickteig, R. ; Hardie, D. G. ; Soderling, Thomas ; Hanley, R. M. ; Kelly, P. T. / Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues. In: European Journal of Biochemistry. 1986 ; Vol. 161, No. 3. pp. 739-747.
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