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CASK (Ca2+, calmodulin-associated serine/threonine kinase) is an essential mammalian cell junction protein and is also crucial at Drosophila neuromuscular synapses. We have shown that CASK is present in mammalian skeletal muscle at the postsynaptic membrane of the neuromuscular junction. CASK interacts biochemically with channels at central synapses, and studies in cultured cells have led to proposed functions for CASK. However, in vivo functions of CASK in skeletal muscle remain unknown. To test hypotheses of CASK functions, we generated two lines of transgenic mice, which overexpress full-length and truncated CASK protein in skeletal muscle. Extensive analyses showed that overexpression of CASK protein did not affect the morphology or physiology of skeletal muscle, the morphology of the neuromuscular junction, or the levels or distribution of protein interactors. These results contrast with previous cell culture experiments and emphasize the importance of in vivo analysis of protein function.

Membrane-associated guanylate kinases (MAGUKs) are cytoplasmic multi-domain proteins that serve as scaffold proteins at cell junctions and synapses. Calmodulin-associated serine/threonine kinase (CASK) stabilizes the integrity of synapses in the brain. Additionally, CASK is capable of acting as a transcriptional co-activator and localizes to neuronal nuclei in the developing brain. We have recently described CASK localization to both the pre- and post-synaptic membranes of the neuromuscular junction (NMJ), where it forms a complex with discs large (Dlg). CASK also localizes to some, but not all nuclei in adult mouse skeletal muscle. To begin to dissect the roles of CASK in the cellular components of the NMJ, we investigated the localization of CASK during differentiation in cell culture models of skeletal muscle and motor neurons. We demonstrate that CASK localizes to the nucleus in undifferentiated myoblasts, but is pre-dominantly in the cytoplasm in differentiated myotubes of the C2C12 myogenic cell line. We also show nuclear localization of both CASK and Dlg in a motor neuron-neuroblastoma hybrid cell line, MN-1, suggesting a role for CASK and Dlg in nuclei of neurons in the peripheral nervous system. Finally, we demonstrate that CASK and Dlg do not co-cluster with acetylcholine receptors in C2C12 myotubes in response to agrin or laminin treatment, suggesting a novel mechanism of recruitment to the NMJ that is independent of acetylcholine receptor and utrophin complexes. These studies delineate important developmental characteristics of CASK and Dlg, and suggest dual roles for these proteins in both the skeletal muscle and motor neuron components of the NMJ., (Copyright 2005 Wiley-Liss, Inc.)