Abstract
Phosphorylation at Threonine 306 and Threonine 307 results in a constitutively inactive CaMKII. This phosphorylation has been shown to be stimulated by CASK-β in low Ca2+ levels and to require binding of CaMKII and CASK-β.1 The CaMKII point mutant T306,7S protein does not interact with CASK-β in vitro. In order to further understand CaMKII and CASK-β interactions, CaMKIIT306,7S and CASK-β null mutant fly lines were created in the Griffith Laboratory (Brandeis University, Waltham MA). CaMKIIT306,7S mutants showed reduced CaMKII activity, while CASK-β-Null mutants showed increased CaMKII activity. Both mutants exhibited locomotion impairments, and the CASK-β null mutant was shown to have reduced ability to release dopamine (DA).2 DA and Tyrosine Hydroxylase (TH), the rate-limiting enzyme in the DA synthetic pathway, were assessed in both mutants in order to further understand how CaMKII and CASK-β impact locomotion. Results indicated that mutation of T306 and T307 to serine downregulates both DA and TH levels. Loss of CASK-β produced an upregulation of DA and TH levels. Genetic rescue experiments done by expression of wild type CaMKII in DArgic neurons of the CaMKIIT306,7S mutants, led to a partial rescue of phenotypes, indicating that the T306,7S mutation is not a gain-of-function mutation. Genetic patterns of expression and molecular function of the CaMKIIT306,7S mutants were assessed through behavioral and mRNA expression assays. Homozygous flies displayed rescue of locomotor behaviors, and mRNA expression showed no significant differences to CaMKIIwt flies. Collectively, our studies support the idea that CASK-β regulates CaMKII through direct interactions, which allows CaMKII to regulate TH activity (either directly or indirectly) and thus affecting the biosynthesis of DA.