The ATP-dependent Membrane Localization of Protein Kinase Cα Is Regulated by Ca²+ Influx and Phosphatidylinositol 4,5-Bisphosphate in Differentiated PC12 Cells

Abstract

Signal transduction through protein kinase Cs (PKCs) strongly depends on their subcellular localization. Here, we investigate the molecular determinants of PKC␣ localization by using a model system of neural growth factor (NGF)-differentiated pheochromocytoma (PC12) cells and extracellular stimulation with ATP. Strikingly, the Ca 2؉ influx, initiated by the ATP stimulation of P2X receptors, rather than the Ca 2؉ released from the intracellular stores, was the driving force behind the translocation of PKC␣ to the plasma membrane. Furthermore, the localization process depended on two regions of the C2 domain: the Ca 2؉ -binding region and the lysine-rich cluster, which bind Ca 2؉ and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ], respectively. It was demonstrated that diacylglycerol was not involved in the localization of PKC␣ through its C1 domain, and in lieu, the presence of PtdIns(4,5)P 2 increased the permanence of PKC␣ in the plasma membrane. Finally, it also was shown that ATP cooperated with NGF during the differentiation process of PC12 cells by increasing the length of the neurites, an effect that was inhibited when the cells were incubated in the presence of a specific inhibitor of PKC␣, suggesting a possible role for this isoenzyme in the neural differentiation process. Overall, these results show a novel mechanism of PKC␣ activation in differentiated PC12 cells, where Ca 2؉ influx, together with the endogenous PtdIns(4,5)P 2 , anchor PKC␣ to the plasma membrane through two distinct motifs of its C2 domain, leading to enzyme activation.

Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+and PIP2Recognition by Its C2 Domain

“…The results presented here further demonstrate that the PKC␣ C2 CBLs, fused to another C2 domain, are sufficient for Ca 2ϩ -dependent translocation to anionic cellular membranes and phospholipid vesicles. However, although previous studies have concluded that the CBLs are responsible for exclusive plasma membrane targeting (Stahelin et al, 2003;Marin-Vicente et al, 2005), the present findings indicate that the intracellular targeting mediated by the PKC␣ C2 CBLs alone is different, and less specific, than the plasma membrane targeting of the native C2 domain. Thus, in the absence of the ␤3-4 hairpin, the CBLs target primarily to the TGN, with minor binding to the plasma membrane still detectable.…”

“…In Vitro Association of Native PKC␣ C2 Domain with Membrane-bound PIP 2 Previous studies have implicated PS and PIP 2 as regulators of PKC␣ C2 domain docking to artificial membranes Stahelin et al, 2003;Marin-Vicente et al, 2005). As stated above, the membrane-bound target of the PLC␦ 1 PH domain, PIP 2 , is specifically enriched in the inner leaflet of the plasma membrane (Roth, 2004), whereas PS is a constituent of all cellular membranes (van Meer, 1998).…”

“…Earlier studies have suggested that lysines 197 and 199 in the ␤3 strand and lysines 209 and 211 in the ␤4 strand of PKC␣C2 are important in Ca 2ϩ -independent, PIP 2 -dependent binding to vesicles, PSand PIP 2 -dependent regulation of PKC␣ activity in vitro, and membrane residence (Ochoa et al, 2002;Corbalan-Garcia et al, 2003;Rodriguez-Alfaro et al, 2004;Marin-Vicente et al, 2005). The hypothesis that these lysine residues are important in PIP 2 recognition predicts that mutations at these positions would weaken membrane-targeting specificity in vivo and lipid-binding affinity in vitro.…”

“…After increases in intracellular Ca 2ϩ concentrations, the PKC␣ C2 domain binds 2 Ca 2ϩ ions in a pocket defined by three interstrand loops (Ca 2ϩ -binding loops or CBLs), leading to a favorable electrostatic interaction with the isolated headgroup of the anionic lipid phosphatidylserine (PS) (Verdaguer et al, 1999) and with PScontaining membranes (Murray and Honig, 2002;Evans et al, 2004). Previous studies have concluded that the CBLs are primarily or fully responsible for specific targeting to the plasma membrane in preference to other intracellular membranes (Stahelin et al, 2003;Marin-Vicente et al, 2005). In addition to the CBLs, a second membrane-interacting region consisting of basic residues in the ␤-hairpin formed by ␤ strands 3 and 4 has been identified and shown to interact with PS or another membrane lipid, PIP 2 (Verdaguer et al, 1999;Ochoa et al, 2002;Corbalan-Garcia et al, 2003).…”

“…In addition to the CBLs, a second membrane-interacting region consisting of basic residues in the ␤-hairpin formed by ␤ strands 3 and 4 has been identified and shown to interact with PS or another membrane lipid, PIP 2 (Verdaguer et al, 1999;Ochoa et al, 2002;Corbalan-Garcia et al, 2003). Mutations at key positions in the CBLs or in the ␤3-4 hairpin interfere with plasma membrane interaction in vivo and reduce the affinity of the PKC␣ C2 domain for PS or PIP 2 in vitro (Medkova and Cho, 1998;Ochoa et al, 2002;Bolsover et al, 2003;Stahelin et al, 2003;Rodriguez-Alfaro et al, 2004;Marin-Vicente et al, 2005).…”