In the present study, we asked the question of which of the PI4K enzymes is important for the generation of the plasma membrane pool of PtdIns4P, the precursor of PtdIns(4,5)P2
during PLC activation in agonist-stimulated cells. This question has not been thoroughly addressed before for lack of appropriate tools to investigate it properly. In a previous study, we showed that Wm-sensitive type III PI4Ks are required for the maintenance of agonist-sensitive phosphoinositide pools and hence Ins(1,4,5)P3
and calcium signaling (Nakanishi et al., 1995
). These studies have been confirmed in other laboratories using different cell lines and different Ca2+
-mobilizing receptors and agonists (Willars et al., 1998
), but the identity of the enzyme(s) responsible for supplying PtdIns4P for the plasma membrane so far has eluded identification. The recent discovery of a PI 3-kinase inhibitor that discriminates between the type III PI4Ks has proven to be an invaluable tool to reinvestigate this question. Based on studies using 32
P-labeled phosphoinositides and myo
H]inositol-labeled inositol phosphates and cytoplasmic Ca2+
measurements, we were able to show that the effects of Wm are reproduced by inhibitors of PI4KIIIα but not PI4KIIIβ. It was also important to demonstrate that the PtdIns4P pool that is affected by the PI4KIIIα enzyme is in fact in the plasma membrane. This question was especially relevant, because none of the type III PI4Ks can be found in detectable amounts in the plasma membrane by immunofluorescence analysis of COS-7 or HEK-293 cells. Based on enzymatic characterization of the plasma membrane-associated PI4K activity performed two decades ago, it was concluded that the smaller and wortmannin-insensitive type II PI4Ks are present in the plasma membrane. This is supported by more recent immunocytochemical analysis that finds only the type II enzymes partially in the plasma membrane. Of the type III enzymes, PI4KIIIβ is found in the Golgi, and PI4KIIIα in the ER/Golgi compartment (see Balla and Balla, 2006
for citation of the original studies). So, it was of particular interest that the PH domain of the OSH2 protein, one of the yeast homologues of oxysterol binding proteins, is capable of reporting on changes in the plasma membrane that are consistent with changes of PtdIns4P levels (see also Yeung et al., 2006
These studies using PH domains of PLCδ1
and OSH2-PH2x to monitor PtdIns(4,5)P2
and PtdIns4P, respectively, confirmed the conclusions of the labeling and Ca2+
experiments that PI4KIIIα is the enzyme necessary for the maintenance of the plasma membrane pool of these lipids. The exact mechanism how this is achieved by the enzyme that is primarily ER/Golgi localized remains to be answered, but its is quite possible that a small fraction of the enzyme is in fact found at the plasma membrane regulated by a unique mechanism, whereas the larger fraction of the protein has additional functions in the ER/Golgi compartments. It is interesting to note that in S. cerevisiae
the Stt4p PI4K, a homologue of PI4KIIIα, was reported to generate the plasma membrane PtdIns4P pool, but unlike in mammalian cells, the yeast enzyme is primarily found in the plasma membrane or a tightly associated compartment (Audhya and Emr, 2002
). Paradoxically, there are well-documented ER and vacuolar functions of Stt4p in yeast; yet, the protein has not been detected in those locations (Trotter et al., 1998
; Audhya et al., 2000
RNAi-mediated depletion of the individual PI4Ks has produced only small effects on 32P-labeled PtdIns4P and PtdIns(4,5)P2 but confirming the identity of the PI4KIIIα enzyme as one responsible for PtdIns4P and PtdIns(4,5)P2 production at the plasma membrane during AngII stimulation. Although the levels of the enzymes can be knocked down to 15–30% of their original values, this level of knockdown produced only small effects in the labeled lipids and none on AngII-induced Ca2+ signaling. This probably reflects the fact that small amounts of these enzymes are sufficient to support the production of this signaling pool of the lipids and cells with more severe knockdown could simply be eliminated or developed adaptive mechanisms to cope with the lack of the protein. This highlights the difficulties with knockdown studies of enzymes of crucial importance and the value of inhibitors that can be tested in short-term experiments.
In recent studies, we were able to show the role of PI4Ks in other aspects of cellular PtdIns4P production where the knockdown data gave as clear results as the pharmacological approach. These included the recruitment of the FAPP1PH domain to the Golgi being mediated by both the PI4KIIIβ and PI4KIIα enzymes (Balla et al., 2005
), and the role of PI4KIIIβ in the ER-to-Golgi transport of ceramide (Toth et al., 2006
). In fact, in the former study we found that PI4KIIIα knockdown was able to decrease the plasma membrane recruitment of the FAPP1PH domain during recovery from a large Ca2+
-mediated PLC activation (Balla et al., 2005
). These studies collectively suggest that PI4Ks probably have multiple functions within the cells that are affected at different level of enzyme depletion.
Last, the experiments using the FAPP1PH, OSBP-PH, and OSH2-PH domains highlight the values and the limitations of PH domain reporters. They clearly demonstrate that specific pools of PtdIns4P are detected by the individual probes and that one reporter may not detect PtdIns4P in all locations within the cells. The FAPP1 and OSBP PH domains detect primarily the Golgi pool, although under special circumstances such as during recovery from massive Ca2+
induced PLC activation they can also detect PtdIns4P in the plasma membrane (Balla et al., 2005
). In contrast, the OSH2–2x-PH-GFP reporter detects PtdIns4P in the plasma membrane but not in the Golgi compartment in mammalian cells. Only the yeast OSH1-PH decorates both the Golgi and the plasma membrane. Importantly, the plasma membrane localization of all of the PtdIns4P reporters depends on the activity of Wm-sensitive, type III PI4Ks, and based on the current studies, specifically of the PI4KIIIα enzyme.
In summary, the present results establish PI4KIIIα as an important component of the generation of the plasma membrane pool of phosphoinositides. This conclusion is based on pharmacological studies and confirmed by RNAi-mediated gene silencing, although the latter could not decrease the enzyme levels sufficiently to appreciably impair Ca2+ signaling. These results also establish the OSH2-PH2x domain as a useful reporter to follow PtdIns4P changes in the plasma membrane. Further studies are in progress to identify the mechanism by which the apparently ER/Golgi localized PI4KIIIα can affect the supply of plasma membrane PtdIns4P.