PHOSPHORYLATION AT THE NUCLEAR-LOCALIZATION SIGNAL OF CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE-II BLOCKS ITS NUCLEAR TARGETING/

Translocation of protein kinases with broad: substrate specificities b etween different subcellular compartments by activation of signaling p athways is an established mechanism to direct the activity of these en zymes toward particular substrates. Recently, we identified two isofor ms of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), whi ch are targeted to the nucleus by an alternatively spliced nuclear loc alization signal (NLS). Here we report that cotransfection with consti tutively active mutants of CaM kinase I or CaM kinase IV specifically blocks nuclear targeting of CaM kinase II as a result of phosphorylati on of a Ser immediately adjacent to the NLS of CaM kinase II. Both CaM kinase I and CaM kinase IV are able to phosphorylate this Ser residue in vitro, and mutagenesis studies suggest that this phosphorylation i s both necessary and sufficient to block nuclear targeting. Furthermor e, we provide experimental evidence that introduction of a negatively charged residue at this phosphorylation site reduces; binding of the k inase to an NLS receptor in vitro, thus providing a mechanism that may explain the blockade of nuclear targeting that me have observed in si tu.