ABSTRACT
Calcium/calmodulin-dependent protein kinase I-alpha (CaMKI-alpha) is a ubiquitous cytosolic enzyme that phosphorylates a number of nuclear proteins in vitro and has been implicated in transcriptional regulation. We report that cytoplasmic localization of CaMKI-alpha depends on CRM1-mediated nuclear export mediated through a Rev-like nuclear export signal in the CaMKI-alpha regulatory domain. Interaction of CaMKI-alpha with a CRM1 complex in vitro is enhanced by incubation with calcium/calmodulin. Translocation of CaMKI-alpha into the nucleus involves a conserved sequence located within the catalytic core. Mutation of this sequence partially blocks nuclear entry of an export-impaired mutant of CaMKI-alpha.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cytoplasm/metabolism , Nuclear Localization Signals/metabolism , Receptors, Cytoplasmic and Nuclear , Active Transport, Cell Nucleus/physiology , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 1 , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Consensus Sequence , Enzyme Activation , Karyopherins , Microscopy, Fluorescence , Nuclear Localization Signals/genetics , PC12 Cells , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Subcellular Fractions/metabolism , Transfection , Exportin 1 ProteinABSTRACT
Much research has focused on pathways leading to opiate addiction. Pathways opposing addiction are more difficult to study but may be critical in developing interventions to combat drug dependence and withdrawal. Galanin decreases firing of locus coeruleus neurons, an effect hypothesized to decrease signs of opiate withdrawal. The current study addresses whether galanin affects morphine withdrawal signs by using a galanin agonist, galnon, that crosses the blood-brain barrier, and mice genetically engineered to under- or overexpress galanin peptide. Galnon significantly decreased morphine withdrawal signs in C57BL/6 mice. Further, knockout mice lacking galanin showed exacerbated morphine withdrawal signs, suggesting that endogenous galanin normally counteracts opiate withdrawal. Transgenic mice overexpressing galanin in noradrenergic neurons also showed decreased morphine withdrawal signs, suggesting a possible neuroanatomical locus for these effects of galanin. Both c-fos immunoreactivity, a marker of neuronal activity, and phosphorylation of tyrosine hydroxylase at Ser-40, a marker of cAMP levels, are decreased in the locus coeruleus by galnon treatment after morphine withdrawal, suggesting a possible molecular mechanism for the behavioral effects of galanin. These studies suggest that galanin normally acts to counteract opiate withdrawal and that small molecule galanin agonists could be effective in diminishing the physical signs of withdrawal.
