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• _6 value "Interestingly, the presence of two additional basic amino acids 6 and 7 residues N-terminal to the phosphorylation site in certain peptide substrates allows phosphorylation by CaMKI/IV equally well with or without activation by a CaMKK (14). This \"activation independence\" has not yet been demonstrated toward protein substrates. Nonetheless, because CaMKI/IV requires Ca2+/CaM for deinhibition in addition to activation loop phosphorylation, substrates of this type would not be phosphorylated by CaMKI/IV until a Ca2+ signal was initiated and so could represent Ca2+-dependent but activation-independent signaling targets. Subsequent activation by a CaMKK would increase the number of available substrates by enhancing CaMKI/IV activity toward a second set of substrates (Fig. 1). Does the Cascade Function in Cells? TOP INTRODUCTION Identification and Biochemical... Does the Cascade Function... Is the Cascade Physiologic? Other Transcriptional Targets... Evidence for a CaMK... REFERENCES The first reconstruction of a CaMK cascade in cells used transient transfection experiments with CREB as the transcriptional target. CaMKI and CaMKIV phosphorylate CREB on its activating Ser-133 in vitro and stimulate Gal4-CREB-dependent transcription in response to a rise in intracellular Ca2+ when cells are cotransfected with Gal4-CREB and a Gal4 reporter gene (15). Additional cotransfection with a CaMKK increases reporter activity more than 10-fold (8, 9). Mutation of the CaMKIV activation loop T to A abolishes CaMKK enhancement. To be a signaling cascade, kinase activation loop phosphorylation must be dependent upon induction of CaMKK activity. Phosphate incorporation into endogenous CaMKIV in Jurkat cells is induced rapidly following T-cell receptor stimulation and is blocked by chelation of extracellular Ca2+. This is accompanied by 8-14-fold increases in immunoprecipitated CaMKIV activity that is refractory to further activation by exogenous CaMKK and can be reversed by in vitro treatment with PP2A (16). Recombinant CaMKIV transfected into BJAB cells, which lack endogenous CaMKIV, demonstrates similar activation following anti-IgM stimulation that is abrogated by mutating the activation loop T to A (4). Likewise, CaMKI phosphorylation is induced in PC12 cells, coincident with increased CaMKI activity and reduced activation of CaMKI by exogenous CaMKK (17). Collectively, these experiments provide strong evidence for an inducible, Ca2+-dependent activation of CaMKI and CaMKIV in intact cells via activation loop phosphorylation. However, the fact that activation loop phosphorylation requires Ca2+ does not confirm that the physiologic activator is itself Ca2+-dependent, because Ca2+/CaM binding to CaMKI or CaMKIV is required before these enzymes can be phosphorylated by the known CaMKKs. The issue of subcellular localization is a confounding one for any model of the CaMK cascade regulating transcription. There are several lines of evidence that CaMKK and - are both cytoplasmic. In contrast to an early study using a polyclonal antibody, immunohistochemistry of rat brain slices using monoclonal antibodies able to distinguish between the and isoforms found exclusively cytoplasmic immunoreactivity for both (18). Furthermore, green fluorescent protein-tagged CaMKK and - are cytoplasmic in NG108 cells even after depolarizing stimulation (18). This holds true for overexpressed CaMKK in Jurkat and BJAB cells.2 Similarly, CaMKI appears to be excluded from the nucleus in brain slices as well as in cells overexpressing the protein, yet CaMKI immunoreactivity has recently been observed to translocate to the nuclei of hippocampal neurons during long term potentiation (19, 20).3 In contrast, CaMKIV is predominantly nuclear but can also be found in neuronal soma and dendritic processes, where it could interact with a cytoplasmic CaMKK (21). The CaMK cascade then is well positioned to affect cytoplasmic e..." provenance.
• _6 wasQuotedFrom 11096122 provenance.