The dependence of calcineurin on Ca2+ for activity is the result of the concerted action of calmodulin, which increases the turnover rate of the enzyme and modulates its response to Ca2+ transients, and of calcineurin B, which decreases the Km of the enzyme for its substrate. The calmodulin-stimulated protein phosphatase calcineurin is under the control of two functionally distinct, but structurally similar, Ca(2+)-regulated proteins, calmodulin and calcineurin B. The Ca(2+)-dependent activation of calcineurin by calmodulin is highly cooperative (Hill coefficient of 2.8-3), and the concentration of Ca2+ needed for half-maximum activation decreases from 1.3 to 0.6 microM when the concentration of calmodulin is increased from 0.03 to 20 microM. Conversely, the affinity of calmodulin for Ca2+ is increased by more than 2 orders of magnitude in the presence of a peptide corresponding to the calmodulin-binding domain of calcineurin A. Calmodulin increases the Vmax without changing the Km value of the enzyme. Unlike calmodulin, calcineurin B interacts with calcineurin A in the presence of EGTA, and Ca2+ binding to calcineurin B stimulates native calcineurin up to only 10% of the maximum activity achieved with calmodulin. The Ca(2+)-dependent activation of a proteolyzed derivative of calcineurin, calcineurin-45, which lacks the regulatory domain, was used to study the role of calcineurin B. Removal of the regulatory domain increases the Vmax of calcineurin, as does binding of calmodulin, but it also increases the affinity of calcineurin for Ca2+. Ca2+ binding to calcineurin B decreases the Km value of calcineurin without changing its Vmax.(ABSTRACT TRUNCATED AT 250 WORDS)

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Protein phosphorylation is involved in nearly all essential biochemical pathways and the deregulation of phosphorylation events has been associated with the onset of numerous diseases. A multitude of tandem mass spectrometry (MS/MS) and multistage MS/MS (i.e., MS(n) ) strategies have been developed in recent years and have been applied toward comprehensive phosphoproteomic analysis, based on the interrogation of proteolytically derived phosphopeptides. However, the utility of each of these MS/MS and MS(n) approaches for phosphopeptide identification and characterization, including phosphorylation site localization, is critically dependant on the properties of the precursor ion (e.g., polarity and charge state), the specific ion activation method that is employed, and the underlying gas-phase ion chemistries, mechanisms and other factors that influence the gas-phase fragmentation behavior of phosphopeptide ions. This review therefore provides an overview of recent studies aimed at developing an improved understanding of these issues, and highlights the advantages and limitations of both established (e.g., CID) and newly maturing (e.g., ECD, ETD, photodissociation, etc.) yet complementary, ion activation techniques. This understanding is expected to facilitate the continued refinement of existing MS/MS strategies, and the development of novel MS/MS techniques for phosphopeptide analysis, with great promise in providing new insights into the role of protein phosphorylation on normal biological function, and in the onset and progression of disease. © 2011 Wiley Periodicals, Inc., Mass Spec Rev 30:600-625, 2011.

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