61] Calmodulin and cold-labile microtubules

Abstract

This chapter discusses an interaction between the activator and heat-stable MAPs, chiefly MAP 2 and the τ proteins that contribute significantly to the regulation of microtubule stability. The two major constituents of microtubules are tubulin and microtubule-associated proteins. A Ca 2+ calmodulin-requiring protein kinase is known to phosphorylate tubulin and especially MAP 2 , and the latter alteration affects the polymerization process. These phosphorylations occur in addition to those produced by cAMP-dependent and tyrosine kinases. Three independent approaches are used to study the interaction of Ca 2+ /calmodulin with the heat-stable MAPs: (1) affinity chromatography on calmodulin columns; (2) cross-linking to MAPS or a MAP fragment; and (3) equilibrium binding using 125 I-labeled calmodulin ([ 125 I]calmodulin). In all cases, the interaction is shown to be Ca 2+ requiring (or EGTA reversed) and sensitive to phenothiazine inhibition. Calmodulin-binding proteins fall into two affinity classes: K d = nanomolar and K d = micromolar. The former are often regulated by Ca 2+ availability, while in the latter class, calmodulin availability is rate limiting as total tissue-calmodulin concentrations are in the neighborhood of the K d for binding to MAP 2 . Cold-stable microtubules from brain show a large calmodulin-stimulated depolymerization effect in the presence of rather large calcium concentrations. The effect is Ca 2+ dependent and occurs at calmodulin concentrations that appear to be stoichiometric with "stop" proteins or MAPs, or both.