Identification of the phosphatidylserine binding site in the C2 domain that is important for PKC alpha activation and in vivo cell localization

The C2 domain of classical PKCs binds to membranes through Ca2+ bridging to phosphatidylserine as recently observed through X-ray diffraction of the i solated domain. Additionally, it has been proposed that N189, T251, R216, a nd R249A interact directly with phosphatidylserine [Verdaguer, N., et al. ( 1999) EMBO J. 18, 6329-6338]. When these four residues were mutated to Ala to determine their role in PKC binding to phospholipid membranes, PKC activ ation, and in its in vivo localization, the results revealed that they were very important for the activation of full-length PKC alpha. N189, in parti cular, was involved in the activation of the enzyme after its interaction w ith PS, since its mutation to Ala did not decrease the level of membrane bi nding but did prevent full enzyme activation. On the other hand, I mutation s R216A, R249A, and T251A affected both membrane binding and enzyme activat ion, although T251A had the most drastic effect, suggesting that the protei n interactions with the carbonyl groups of the phospholipid are also a key event in the activation process. Taken together, these results show that th e four residues located near the calcium binding site are critical in phosp hatidylserine-dependent PKCa activation, in which N189 plays an important r ole, triggering the enzyme activation probably by interacting with neighbor ing residues of the protein when lipid binding occurs. Furthermore, these r esults provide strong evidence for better defining one of the two phosphati dylserine isomer models proposed in the previous crystallographic report.