Presence in many mammalian tissues of an identical major cytosolic substrate (Mr 100000) for calmodulin‐dependent protein kinase

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Abstract
Incubation of cytosol fractions from a variety of mammalian tissues (heart, liver, lung, adrenal, spleen and skeletal muscle) with Ca2+ (0.5 mM) in the presence of γ‐[32P]ATP resulted in the phosphorylation of a prominent substrate of M r ∼ 100000 (100 kDa). One‐dimensional peptide maps and two‐dimensional tryptic fingerprints of the phosphoprotein from these sources were identical. A single major phosphopeptide was generated by trypsin and was determined to contain exclusively phosphothreonine. The 100 kDa substrate could be distinguished from glycogen phosphorylase (M r ∼ 97000) by a number of criteria including phosphopeptide mapping and by its failure to bind either to glycogen or to a specific antiphosphorylase antibody. The Ca2+‐dependent protein kinase responsible for phosphorylation of the 100 kDa protein appeared to be a calmodulin (CaM)‐requiring enzyme in that it could be inhibited in cytosol extracts by trifluoperazine (IC 50 6–16 μM) and that exogenous CaM was necessary for 100 kDa phosphorylation in CaM‐depleted cytosol. These results suggest that a rise in intracellular Ca2+ resulting in an activation of CaM‐dependent protein kinase leads to the phosphorylation of a common 100 kDa substrate in many tissues.