Regulation of the CaMKIV/PP2A Signaling Module
Reece, Kelie M'liss
Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine kinase that plays a crucial role in the control of synaptic plasticity and T cell maturation. Activation of CaMKIV requires Ca2+/calmodulin binding and phosphorylation at T200 by CaMK kinase. Previous work from my laboratory showed that protein serine/threonine phosphatase 2A (PP2A) forms a complex with CaMKIV, and negatively regulates the phosphorylation state and activity of the kinase. My thesis studies focused on understanding the molecular mechanisms underlying the regulation and assembly of the CaMKIV•PP2A complex. I demonstrate, using a novel CaMKIV phospho-specific antibody, that PP2A negatively regulates CaMKIV by directly dephosphorylating the kinase on its phospho-Thr200 residue within the kinase. In addition, I show that PP2A regulates the endogenous kinase in a tight and acute manner, but has little effect on the ectopically-expressed kinase. This differential regulation of endogenous versus ectopic CaMKIV by PP2A does not appear to be due to differences in the subcellular localization of the kinase, as both forms of the kinases exhibited similar subcellular distribution profiles. Rather, the differences of endogenous versus ectopic CaMKIV regulation by PP2A is due to the fact that the PP2A catalytic subunit (PP2Ac) binds more efficiently to the endogenous enzyme. However, overexpression of either the B or Bregulatory subunit of PP2A causes the recruitment of PP2Ac to the ectopic CaMKIV, thus facilitating the assembly of a CaMKIV•PP2A complex. These B- and B-containing holoenzymes also preferentially dephosphorylate CaMKIV in vitro, thus indicating that they may be the primary modulators of CaMKIV in cells. Together, my findings provide new insights into the regulation of CaMKIV by an associated PP2A holoenzyme, and lay the foundation for future studies aimed at uncovering novel aspects of the biology of this key signaling module.