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Calmodulin's calculated electrostatic potential surface is asymmetrically distributed about the molecule. Concentrations of uncompensated negative charge are localized near certain alpha-helices and calcium-binding loops. Further calculations suggest that these charge features of calmodulin can be selectively perturbed by changing clusters of phylogenetically conserved acidic amino acids in helices to lysines. When these cluster charge reversals are actually produced by using cassette-based site-specific mutagenesis of residues 82-84 or 118-120, the resulting proteins differ in their interaction with two distinct calmodulin-dependent protein kinases, myosin light chain kinase and calmodulin-dependent protein kinase II. Each calmodulin mutant can be purified to apparent chemical homogeneity by an identical purification protocol that is based on conservation of its overall properties, including calcium binding. Although cluster charge reversals result in localized perturbations of the computed negative surface, single amino acid changes would not be expected to alter significantly the distribution of the negative surface because of the relatively high density of uncompensated negative charge in the region around residues 82-84 and 118-120. However, this does not preclude the possibility of single amino acid charge perturbations having a functional effect on the more intimate, catalytically active complex. The electrostatic surface of calmodulin described in this report may be a feature that would be altered only by cluster charge reversal mutations. Overall, the results suggest that the charge properties of calmodulin are one of several properties that are important for the efficient assembly of calmodulin-protein kinase signal transduction complexes in eukaryotic cells.

The Ca2 +/calmodulin (CaM)-dependent protein phosphatase calcineurin is rapidly phosphorylated (0.8 mol of 32PO4 per mol of 60-kDa subunit ofcalcineurin) by brain Ca2+/CaM-dependent protein kinase H (CaM-kinase II). This reaction requires the autophosphorylated, Ca2+-independent form of CaM-kinase H since Ca2 +/CaM binding to calcineurin inhibits phosphorylation. However, the phosphorylation reaction does require Ca2 , presumably acting through the 19-kDa subunit of calcineurin. Calcineurin is a good substrate for The observation that addition of EGTA to the partially Ca2 -independent form of CaM-kinase II stimulated further autophosphorylation suggests that this site of Ca2+-independent autophosphorylation may be masked by the binding ofCa2 +/CaM. This interpretation would be consistent with the fact that after the Ca2"-independent autophosphorylation the kinase is no longer stimulated by Ca2`/CaM (11). Examination of the amino acid sequence of the 50-kDa (12, 13) and 60-kDa (14) subunits of CaM-kinase II shows two consensus phosphorylation sites flanking the CaM-binding domain (12)(13)(14) that would be likely candidates for this autophosphorylation. Precedent exists for inhibition of phosphorylation through binding of Ca2+/CaM. For example, it is known that binding of CaM to smooth muscle myosin light chain kinase blocks the phosphorylation of one of the two sites by other protein kinases (15-17).Based on these precedents, we wondered whether other CaM-binding proteins may also have phosphorylation sites that are masked by binding of Ca2 +/CaM and would therefore only be phosphorylated by the Ca2"-independent form of CaM-kinase II. If this concept was correct, it would suggest an additional regulatory consequence of autophosphorylation of CaM-kinase II: the existence of a set of phosphorylation sites only available at basal levels of Ca2+ to the Ca2'-independent form of CaM-kinase II. We chose to first examine the Ca2+/CaM-dependent protein phosphatase calcineurin (CaN), since purified CaN does contain phosphate (up to 0.6 7001The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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