The C2 domain of classical PKCs binds to membranes through Ca(2+) bridging to phosphatidylserine as recently observed through X-ray diffraction of the isolated domain. Additionally, it has been proposed that N189, T251, R216, and 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 activation, and in its in vivo localization, the results revealed that they were very important for the activation of full-length PKCalpha. N189, in particular, was involved in the activation of the enzyme after its interaction with PS, since its mutation to Ala did not decrease the level of membrane binding but did prevent full enzyme activation. On the other hand, mutations R216A, R249A, and T251A affected both membrane binding and enzyme activation, although T251A had the most drastic effect, suggesting that the protein interactions with the carbonyl groups of the phospholipid are also a key event in the activation process. Taken together, these results show that the four residues located near the calcium binding site are critical in phosphatidylserine-dependent PKCalpha activation, in which N189 plays an important role, triggering the enzyme activation probably by interacting with neighboring residues of the protein when lipid binding occurs. Furthermore, these results provide strong evidence for better defining one of the two phosphatidylserine isomer models proposed in the previous crystallographic report.