The proposed studies will investigate the mechanism of activation of Ca++/calmodulin-dependent protein kinase IV (CaM kinase IV) during hypoxia that leads to activation of cyclic AMP-response element binding (CREB) protein and triggers the transcription of apoptotic genes resulting in hypoxic neuronal death. We will test the hypothesis that the mechanism of increased activation of CaM kinase IV during hypoxia is due to tyrosine phosphorylation of calmodulin and tyrosine phosphorylation of CaM kinase IV by the tyrosine kinases, Src tyrosine kinase and epidermal growth factor receptor (EGFR) tyrosine kinase. We propose that cerebral hypoxia-induced increased tyrosine phosphorylation of calmodulin (at Tyr99) leads to increased affinity for its binding at the calmodulin binding domain of the CaM kinase IV enzyme and results in increased activation of CaM kinase IV. The increased tyrosine phosphorylation of CaM kinase IV (at Tyr154 and Tyr172) leading to modification of the active site domain of the enzyme leads to increased affinity for its substrate and results in increased activation of CaM kinase IV. Experimental protocols will be carried out on newborn piglets investigating: (1) the effect of hypoxia on Tyr99 phosphorylation of calmodulin, Tyr154 and Tyr172 phosphorylation of CaM kinase IV, and activity of CaM kinase IV in neuronal nuclei, (2) the effect of hypoxia on the activity of Src kinase and EGFR kinase, and tyrosine phosphorylation at the active site of Src kinase (Tyr416) and EGFR kinase (Tyr1173), an index of enzyme activation, using specific anti-Tyr416-p-Src kinase and anti-Tyr1173-p-EGFR kinase antibodies to demonstrate activation of Src and EGFR tyrosine kinases in the cerebral cortex of hypoxic piglets.
The proposed studies will establish a novel mechanism of Src and EGFR kinase-dependent activation of CaM kinase IV that triggers the transcription of pro-apoptotic genes. The elucidation of the mechanism of activation of CaM kinase IV during hypoxia will enable us to develop Src tyrosine kinase and EGFR tyrosinekinase inhibition-based therapeutic strategies to prevent hypoxia-induced brain injury in the newborn.
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