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Phospholipase C (PLC)delta1 shuttles between the nucleus and the cytoplasm. Here, we demonstrate that treatment of MDCK cells and PC12 cells with ionomycin causes nuclear accumulation of ectopically expressed and endogenous PLCdelta1, respectively, suggesting that signals that increase [Ca2+]i trigger nuclear translocation. To clarify the molecular mechanisms involved in this translocation, we have examined whether PLCdelta1 binds with importins. PLCdelta1 interacted with importin beta1 in a Ca2+-dependent manner in vitro even in the absence of importin alpha. A PLCdelta1 mutant E341A, which lacks Ca2+-binding to the catalytic core, did not show this interaction at any physiological Ca2+ concentration and did not translocate into the nucleus after ionomycin treatment when expressed in MDCK cells. These results suggested that the nuclear import of PLCdelta1 is mediated by its Ca2+-dependent interaction with importin beta1.

Phospholipase C (PLC) activation in neonatal rat ventricular cardiomyocytes (NRVM) generates inositol(1,4,5)trisphosphate (Ins(1,4,5)P(3)) in response to elevations in Ca(2+) or inositol(1,4)bisphosphate in response to G protein stimulation. Overexpression of PLCdelta(1) increased total [(3)H]inositol phosphate (InsP) content and elevated [(3)H]Ins(1,4,5)P(3), but failed to increase [(3)H]InsP responses to the Ca(2+) ionophore A23187. Antisense PLCdelta(1) expression reduced endogenous PLCdelta(1) content but did not decrease the A23187 response. In permeabilized NRVM, [(3)H]InsP responses to elevated Ca(2+) were not inhibited by Ins(1,4,5)P(3), even at concentrations 1000-fold greater than required for selective inhibition of PLCdelta(1). Taken together these data provide evidence that PLCdelta(1) does not mediate the InsP response to elevated Ca(2+) in NRVM.

Since their discovery almost 10 years ago pleckstrin homology (PH) domains have been identified in a wide variety of proteins. Here, we focus on two proteins whose PH domains play a defined functional role, phospholipase C (PLC)-beta(2) and PLCdelta(1). While the PH domains of both proteins are responsible for membrane targeting, their specificity of membrane binding drastically differs. However, in both these proteins the PH domains work to modulate the activity of their catalytic core upon interaction with either phosphoinositol lipids or G protein activators. These observations show that these PH domains are not simply binding sites tethered onto their host enzyme but are intimately associated with their catalytic core. This property may be true for other PH domains.

Pleckstrin homology (PH) domains are protein modules found in proteins involved in many cellular processes. The majority of PH domain-containing proteins require membrane association for their function. It has been shown that most PH domains interact directly with the cell membrane by binding to phosphoinositides with a broad range of specificity and affinity. While a highly specific binding of the PH domain to a phosphoinositide can be necessary and sufficient for the correct recruitment of the host protein to the membrane, a weaker and less specific interaction may be necessary but not sufficient, thus probably requiring alternative, co-operative mechanisms.