Biochemical characterization and localization of the dual specificity kinase CLK1

CLK1 was one of the first identified dual specificity kinases and is the fo unding member of the 'LAMMER' family of kinases, We have established the su bstrate site specificity of CLK1, We report here that truncation of the N t erminus 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 th at this domain does not contain a pseudo-substrate motif, but may act to co nformationally constrain the catalytic activity of CLK1. Tyrosine phosphory lation has been proposed to be critical for CLK1 activity, however, CLK1 ac tivity was unaffected by exposure to tyrosine phosphatases. Treatment of CL K1 with the serine/threonine specific phosphatase PP2A, resulted in a 2- to 6-fold increase in enzymatic activity, Incubation of CLK1 with tyrosine ph osphatases in combination with PP2A abolished CLK1 activity, These data sug gest that CLK1 is regulated by three distinct mechanisms that serve to both positively and negatively regulate CLK1 activity, CLK1 activity is positiv ely regulated by phosphorylation on either tyrosine residues or serine/thre onine residues, and is negatively regulated by steric constraints mediated by the N-terminal domain, as well as, by phosphorylation on a subset of ser ine/threonine residues within the catalytic domain, CLK1 mRNA is expressed at low levels in all tissues and cell lines examined. The full-length and t runcated splice forms are expressed at roughly equivalent levels in most ti ssues, The ratio of the two splice variants of CLK1 can be altered by treat ment with cycloheximide. CLK1 protein expression is limited to a small subs et of highly localized neuronal populations in the rat brain, Contrary to p revious studies using overexpression systems, me show that CLK1 protein is primarily found in the cytoplasm of these cells, with only a small fraction localized to the nucleus.