Analysis of CaM-kinase signaling in cells.

A change in intracellular free calcium is a common signaling mechanism that modulates a wide array of physiological processes in most cells. Responses to increased intracellular Ca2+ are often mediated by the ubiquitous protein calmodulin (CaM) that upon binding Ca2+ can interact with and alter the functionality of numerous proteins including a family of protein kinases referred to as CaM-kinases (CaMKs). Of particular interest are multifunctional CaMKs, such as CaMKI, CaMKII, CaMKIV and CaMKK, that can phosphorylate multiple downstream targets. This review will outline several protocols we have used to identify which members and/or isoforms of this CaMK family mediate specific cellular responses with a focus on studies in neurons. Many previous studies have relied on a single approach such as pharmacological inhibitors or transfected dominant-negative kinase constructs. Since each of these protocols has its limitations, that will be discussed, we emphasize the necessity to use multiple, independent approaches in mapping out cellular signaling pathways.