Biochemical characterization and localization of the dual specificity kinase CLK1.

CLK1 was one of the first identified dual specificity kinases and is the founding member of the 'LAMMER' family of kinases. We have established the substrate site specificity of CLK1. We report here that truncation of the N terminus of CLK1 resulted in a dramatic increase in CLK1 enzymatic activity, indicating that the N terminus acts as a negative regulatory domain. The N-terminal truncation resulted in a 45-fold increase in V(max), suggesting that this domain does not contain a pseudo-substrate motif, but may act to conformationally constrain the catalytic activity of CLK1. Tyrosine phosphorylation has been proposed to be critical for CLK1 activity, however, CLK1 activity was unaffected by exposure to tyrosine phosphatases. Treatment of CLK1 with the serine/threonine specific phosphatase PP2A, resulted in a 2- to 6-fold increase in enzymatic activity. Incubation of CLK1 with tyrosine phosphatases in combination with PP2A abolished CLK1 activity. These data suggest that CLK1 is regulated by three distinct mechanisms that serve to both positively and negatively regulate CLK1 activity. CLK1 activity is positively regulated by phosphorylation on either tyrosine residues or serine/threonine residues, and is negatively regulated by steric constraints mediated by the N-terminal domain, as well as, by phosphorylation on a subset of serine/threonine residues within the catalytic domain. CLK1 mRNA is expressed at low levels in all tissues and cell lines examined. The full-length and truncated splice forms are expressed at roughly equivalent levels in most tissues. The ratio of the two splice variants of CLK1 can be altered by treatment with cycloheximide. CLK1 protein expression is limited to a small subset of highly localized neuronal populations in the rat brain. Contrary to previous studies using overexpression systems, we show that CLK1 protein is primarily found in the cytoplasm of these cells, with only a small fraction localized to the nucleus.