Spatial Patterns of Ca2+ Signals Define Intracellular Distribution of a Signaling by Ca2+/Calmodulin-dependent Protein Kinase II

Abstract

Cell signaling is the fundamental strategy by which cells respond to extracellular stimuli. Intracellular distribution of cell signaling is considered to be an important factor affecting the manner in which cells respond to extracellular stimuli with spatial specificity (1, 2). Although little is known of the spatial aspect of cell signaling, that of Ca 2ϩ signaling visualized by Ca 2ϩ microscopy is presently the best characterized example. Numbers of reports have shown that intracellular Ca 2ϩ signals occur locally and globally (3-6). The intracellular distribution of Ca 2ϩ signaling in various types of cells was defined by the amplitude and direction of extracellular stimuli (7-10). Therefore, it has been speculated that spatial patterns of Ca 2ϩ signals might be transmitted, as a form of cellular information, by a downstream molecule that induces Ca 2ϩ -dependent cellular responses (1-2, 6 -10).To address this issue, we visualized site-specific phosphorylation of vimentin by CaMKII 1 and Ca 2ϩ signaling in the same astrocytes. CaMKII is located downstream of Ca 2ϩ signaling and is thought to regulate various cellular responses (11,12). Vimentin is an intermediate filament protein distributed widely in the cytoplasm (13,14) and is phosphorylated by several protein kinases, including CaMKII, in vivo (15, 16). Therefore, vimentin can serve as a substrate for the examination of the cytoplasmic distribution of protein kinase activities (17, 18). Here we report that vimentin phosphorylation by CaMKII was induced locally and globally by Ca 2ϩ signaling. The intracellular area of the phosphorylation was precisely defined by that of Ca 2ϩ signaling. EXPERIMENTAL PROCEDURESPreparation of Antibodies, Peptides, and Proteins-Production of monoclonal antibodies YT33, TM50, 4A4, and MO82 was reported elsewhere (19 -21). Vimentin peptides PV6 (Cys-Ser-Thr-Arg-Ser-Val-phosphoSer 6 -Ser-Ser-Ser-Tyr-Arg), V6 (Cys-Ser-Thr-Arg-Ser-Val-Ser-SerSer-Ser-Tyr-Arg), PV38 (Cys-Ser-Thr-Arg-Thr-Tyr-phosphoSer 38 -LeuGly-Ser-Ala-Leu), and V38 (Cys-Ser-Thr-Arg-Thr-Tyr-Ser-Leu-Gly-SerAla-Leu) were synthesized as described previously (21). A monoclonal antibody against PV6 (MO6) and a polyclonal antibody against PV38 (GK38) were produced following the methods described previously (21, 22) Then the specificity of MO6 and GK38 was checked by enzymelinked immunosorbent assay (21). MO6 bound to PV6 but not to the unphosphorylated peptide V6, while GK38 reacted with PV38 but not with the unphosphorylated form V38. Production of recombinant vimentin and vimentin phosphorylated by CaMKII was described previously (19). The affinity-purified antibody specific for both 50-and 60-kDa subunits of CaMKII (23) was provided by Drs. K. Fukunaga and E. Miyamoto (Kumamoto University).Cell Preparation and Drug Application-Primary cultured astrocytes (type 1 astrocytes) were prepared from the cerebral cortices of newborn rats as described previously (19). Two days before the experiments, astrocytes were subcultured on collagen (type 1, Sigma)-coated glass coversl...

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