Mechanism of CaM kinase IV activation during hypoxia in neuronal nuclei of the cerebral cortex of newborn piglets: the role of Src kinase.

The present study aims to investigate the mechanism of CaM kinase IV activation during hypoxia and tests the hypothesis that hypoxia-induced increased activity of CaM kinase IV is due to Src kinase mediated increased tyrosine phosphorylation of calmodulin and CaM kinase IV in neuronal nuclei of the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, F(i)O(2) of 0.07 for 1 h, n = 5) and hypoxic-pretreated with Src kinase inhibitor PP2 (Hx-Srci, n = 5) groups. Src inhibitor was administered (1.0 mg/kg, I.V.) 30 min prior to hypoxia. Neuronal nuclei were isolated and purified, and tyrosine phosphorylation of calmodulin (Tyr(99)) and CaM kinase IV determined by Western blot using anti-phospho-(pTyr(99))-calmodulin, anti-pTyrosine and anti-CaM kinase IV antibodies. The activity of CaM kinase IV and its consequence the phosphorylation of CREB protein at Ser(133) were determined. Hypoxia resulted in increased tyrosine phosphorylation of calmodulin at Tyr(99), tyrosine phosphorylation of CaM kinase IV, activity of CaM kinase IV and phosphorylation of CREB protein at Ser(133). The data show that administration of Src kinase inhibitor PP2 prevented the hypoxia-induced increased tyrosine phosphorylation of calmodulin (Tyr(99)) and tyrosine phosphorylation of CaM.kinase IV as well as the activity of CaM kinase IV and CREB phosphorylation at Ser(133). We conclude that the mechanism of hypoxia-induced increased activation of CaM kinase IV is mediated by Src kinase-dependent tyrosine phosphorylation of the enzyme and its activator calmodulin. We propose that Tyr(99) phosphorylated calmodulin, as compared to non-phosphorylated, binds with a higher affinity at the calmodulin binding site (rich in basic amino acids) of CaM kinase IV leading to increased activation of CaM kinase IV. Similarly, tyrosine phosphorylated CaM kinase IV binds its substrate with a higher affinity and thus increased tyrosine phosphorylation leads to increased activation of CaM kinase IV resulting in increased CREB phosphorylation that triggers increased transcription of proapoptotic proteins that initiate hypoxic neuronal death.