Autophosphorylation of multifunctional Ca*+/calmodulin-dependent protein kinase converts it from a Ca2+-dependent to a Ca2+-independent or autonomous kinase, a process that may underlie some long-term enhancement of transient Caz+ signals. We demonstrate that the neuronal a subunit clone expressed in COS-7 cells (a-CaM kinase) is sufficient to encode the regulatory phenomena characteristic of the multisubunit kinase isolated from brain. Activity of a-CaM kinase is highly dependent on CaVcalmodulin. It is converted by autophosphorylation to an enzyme capable of Ca2+-independent (autonomous) substrate phosphorylation and autophosphorylation. Using site-directed mutagenesis, we separately eliminate five putative autophosphorylation sites within the regulatory domain and directly examine their individual roles. Ca2+/calmodulin-dependent kinase activity is fully retained by each mutant, but Thrzas is unique among the sites in being indispensable for generation of an autonomous kinase.

Intracellular Ca2+ is an important second messenger in neuronal tissue mediating both immediate and longterm effects of numerous neurotransmitters and neuromodulators. A prominent neuronal enzyme mediating the effects of Ca2+ is multifunctional Caz+/calmodulindependent protein kinase (multifunctional CaM kinase). The importance of multifunctional CaM kinase is attested to by its broad range of substrates, which includes synapsin I, tyrosine hydroxylase, pyruvate kinase, glycogen synthase, microtubule-associated protein 2 (MAPZ), and tau (reviewed in Kennedy et al., 1987; Schulman, 1988; Schulman and Lou, 1989). While it is found in diverse tissues, it is most prominent in neuronal tissue, where it has been estimated to constitute 0.25%-0.5% of total rat brain protein and as much as 2% of hippocampal protein (Erondu and Kennedy, 1985). Multifunctional CaM kinase from rat brain consists of homologous a (54 kd), B (60 kd), p’(58 kd), and y (59 kd) subunits in different isozymic ratios. There is both regional and developmental regulation of the subunit ratio (McGuinness et al., 1985; Miller and Kennedy, 1985;