CaM-kinase II dephosphorylates Thr(286) by a reversal of the autophosphorylation reaction

Autophosphorylation of CaM-kinase II produces a form of the enzyme not requ iring Ca2+/calmodulin for sustained activity. We report that autophosphoryl ated CaM-kinase II dephosphorylates itself in the presence of ADP (termed a utodephosphorylation). The dephosphorylation was unaffected by phosphatase inhibitors and was nucleotide specific, occurring with ADP but not with AMP , GTP, or GDP. P-32-ATP was produced when ADP was added to P-32-labeled CaM -kinase II, indicating that the enzyme was undergoing dephosphorylation thr ough a reversal of the autophosphorylation reaction. ATP addition also prod uced loss of P-32 from the autophosphorylated enzyme while maintaining the kinase in a phosphorylated state. This indicates that the enzyme was underg oing cycles of autophosphorylation and dephosphorylation in the activated s tate. Autothiophosphorylated CaM-kinase II was resistant to autodephosphory lation, Site-directed mutants were used to show that Thr(286) was the predo minant site dephosphorylated, Additionally, CaM-kinase II composed of beta subunits exhibited autodephosphorylation. Ca2+/CaM-independent activity exp ressed by the autophosphorylated alpha and beta holoenzymes was reversed fo llowing autodephosphorylation. (C) 2001 Academic Press.