The CDC42 small GTPase is a major influence on actin-myosin cytoskeleton organization and dynamics, signalling via effector proteins including the Myotonic dystrophy related CDC42-binding protein kinases (MRCK) a and p. We previously identified Serine1003 of MRCKa as a site of autophosphorylation, and showed that a phosphorylation-sensitive antibody raised against this site could be used as a surrogate indicator of kinase activity. In this study, a kinase-dead version of MRCKp was established by mutation of the conserved Lysine 105 to Methionine (K105M), which was then used for mass spectrometry analysis to identify phosphorylation events that occurred in catalytically-competent MRCKp but not in the kinase-dead form. A total of ten phosphorylationswere identified on wild-type MRCKp, of which the previously undescribed Threonine 1108 (Thr1108) was not found on kinase-dead MRCKp K105M, consistent with this being due to autophosphorylation. Mutation of Thr1108 to non-phosphorylatable Alanine (T1108A) or phosphomimetic Glutamate (T1108E) did not affect the ability of MRCKp to phos-phorylate recombinant myosin light chain in vitro, or observably alter the subcellular localization of green fluorescent protein (GFP)-tagged MRCKp expressed in MDA MB 231human breast cancer cells. Although phosphorylation of Thr1108 did not appear to contribute to MRCKp function or regulation, the identification of this phosphorylation does make it possible to characterize whether this site could be used as a surrogatebiomarker of kinase activity and inhibitor efficacy as we previously demonstrated for Ser 1003 in MRCKa.
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