In view of the interest shown in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) as a second messenger, we studied the activation of protein kinase C␣ by this phosphoinositide. By using two double mutants from two different sites located in the C2 domain of protein kinase C␣, we have determined and characterized the PtdIns(4,5)P 2 -binding site in the protein, which was found to be important for its activation. Thus, there are two distinct sites in the C2 domain: the first, the lysinerich cluster located in the ␤3-and ␤4-sheets and which activates the enzyme through direct binding of PtdIns(4,5)P 2 ; and the second, the already well described site formed by the Ca 2؉ -binding region, which also binds phosphatidylserine and a result of which the enzyme is activated. The results obtained in this work point to a sequential activation model, in which protein kinase C␣ needs Ca 2؉ before the PtdIns(4,5)P 2 -dependent activation of the enzyme can occur.Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 1 plays a key role in phosphoinositide signaling and regulates a wide range of processes at many subcellular sites. It is primarily detected in the plasma membrane but is also found in secretory vesicles, lysosomes, in the endoplasmic reticulum, the Golgi, and in the nucleus (1-5). PtdIns(4,5)P 2 can either bind to intracellular proteins and directly modulate their subcellular localization and activity, or it can act as a precursor for the generation of different second messengers. For example, several families of phospholipase C enzymes are responsible for the hydrolysis of PtdIns(4,5)P 2 in cells, leading to the production of diacylglycerol and inositol 1,4,5-trisphosphate (4, 6), which may, in turn, lead to the activation of different proteins such as some PKC isotypes.Protein kinase C (PKC) composes a large family of serine/ threonine kinases, which is activated by many extracellular signals and plays a critical role in many signal-transducing pathways in the cell (7-9). Based on their enzymatic properties, the mammalian PKC isotypes have been grouped into smaller subfamilies. The first group, which includes the classical isoforms ␣, ␤I, ␤II, and ␥, can be distinguished from the other groups because its activity is regulated by diacylglycerol (DAG) and, cooperatively, by Ca 2ϩ and acidic phospholipids, particularly phosphatidylserine (PS). Members of the second group are the novel mammalian (␦, ⑀, , and ) and yeast PKCs that are not regulated by Ca 2ϩ . The third group comprises the atypical PKC isoforms, , , and , whose regulation has not been clearly established, although it is clear that they are not regulated by DAG or Ca 2ϩ (8, 10). In classical PKC isoenzymes, Ca 2ϩ -dependent binding to membranes shows a high specificity for 1,2-sn-phosphatidyl-Lserine (11)(12)(13)(14). Additionally, this group of isoenzymes is sensitive to other anionic phospholipids, including phosphatidic acid and polyphosphoinositides (15-16) and to a variety of amphipathic membrane compounds, such as arachidonic acid and fre...

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Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural and thermal denaturation of the C2 domain of PKC alpha (PKC-C2) and its complexes with Ca(2+) and phosphatidic acid vesicles. The amide I regions in the original spectra of PKC-C2 in the Ca(2+)-free and Ca(2+)-bound states are both consistent with a predominantly beta-sheet secondary structure below the denaturation temperatures. Spectroscopic studies of the thermal denaturation revealed that for the PKC-C2 domain alone the secondary structure abruptly changed at 50 degrees C. While in the presence of 2 and 12.5 mM Ca(2+), the thermal stability of the protein increased to 60 and 70 degrees C, respectively. Further studies using a mutant lacking two important amino acids involved in Ca(2+) binding (PKC-C2D246/248N) demonstrated that these mutations were inherently more stable to thermal denaturation than the wild-type protein. Phosphatidic acid binding to the PKC-C2 domain was characterized, and the lipid-protein binding became Ca(2+)-independent when 100 mol% phosphatidic acid vesicles were used. The mutant lacking two Ca(2+) binding sites was also able to bind to phosphatidic acid vesicles. The effect of lipid binding on secondary structure and thermal stability was also studied. Beta-sheet was the predominant structure observed in the lipid-bound state, although the percentage represented by this structure in the total area of the amide I band significantly decreased from 60% in the lipid-free state to 47% in the lipid-bound state. This decrease in the beta-sheet component of the lipid-bound complex correlates well with the significant increase observed in the 1644 cm(-1) band which can be assigned to loops and disordered structure. Thermal stability after lipid binding was very high, and no sign of thermal denaturation was observed in the presence of lipids under the conditions that were studied.

PKCepsilon is a member of the group of novel PKCs that contain a C2 domain located in their N-terminal region. On the basis of recent structural studies, a series of mutants were prepared to increase our knowledge of the mechanism of the phospholipid binding site of this domain. The results revealed that this domain preferentially binds to phosphatidic acid- and phosphatidylserine-containing vesicles. Although the increase in affinity was linear in the case of phosphatidic acid, it became exponential when the vesicles contained increasing concentrations of phosphatidylserine. Site-directed mutagenesis studies showed that residues W23, R26, and R32 located in loop 1 and I89 and Y91 located in loop 3 are of critical importance when the binding is performed with phosphatidic acid-containing vesicles. Furthermore, when the same mutants were assayed with phosphatidylserine-containing vesicles, no binding was observed in any case, reflecting the smaller affinity of the C2 domain for phosphatidylserine-containing vesicles. A study of the ionic nature of the membrane interaction suggested that it is mainly driven by electrostatic interactions that are disrupted by very low salt concentrations. Differential scanning calorimetry experiments performed to ascertain whether this interaction affected the transition phase of the phosphatidic acid demonstrated that increasing concentrations of the protein lead to changes in the transition, with more than one peak appearing at lower temperatures, which suggests a weak interaction focused on the polar headgroup of the phospholids. In conclusion, a different membrane-binding mode from those previously described in other C2 domains has been found and is seemingly based on electrostatic, interfacial, and hydrophobic interactions without the participation of Ca(2+) ions.

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