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 CKII? subunit. This PLD-induced CKII? degradation was mediated by ubiquitin-proteasome machinery, but MAP kinase and mTOR were not involved in CKII? degradation. PLD isozymes interacted with the CKII? subunit. Immunocytochemical staining revealed that PLD and CKII? colocalize in the cytoplasm of NIH3T3 cells, especially in the perinuclear region. PLD binding to CKII? inhibited CKII? autophos-phorylation, which is known to be important for CKII? stability. In summary, the current data indicate that PLD isozymes can down-regulate CKII activity through the acceleration of CKII beta degradation by ubiquitin-proteasome machinery.

Keywords: CKII regulation, NIH3T3 cells, PLD, protein-protein interaction, ubiquitin-proteasome