Over-expression of phospholipase D isozymes down-regulates protein kinase CKII activity via proteasome-dependent CKIIbeta degradation in NIH3T3 cells.

Abstract

Over-expression of phospholipase D (PLD) 1 or PLD2 down-regulated CKII activity in NIH3T3 cells. The same results were found with catalytically inactive mutants of PLD isozymes, indicating that the catalytic activity of PLD is not required for PLD-mediated CKII inhibition. Consistent with this, 1-butanol did not alter CKII activity. The reduction in CKII activity in PLD-over-expressing NIH3T3 cells was due to reduced protein level, but not mRNA level, of the CKIIbeta subunit. This PLD-induced CKIIbeta degradation was mediated by ubiquitin-proteasome machinery, but MAP kinase and mTOR were not involved in CKIIbeta degradation. PLD isozymes interacted with the CKIIbeta subunit. Immunocyto-chemical staining revealed that PLD and CKIIbeta colocalize in the cytoplasm of NIH3T3 cells, especially in the perinuclear region. PLD binding to CKIIbeta inhibited CKIIbeta autophosphory-lation, which is known to be important for CKIIbeta stability. In summary, the current data indicate that PLD isozymes can down-regulate CKII activity through the acceleration of CKIIbeta degradation by ubiquitin-proteasome machinery.