The C2 domain is a Ca 2؉ -dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. C2 domains are unique among membrane targeting domains in that they show a wide range of lipid selectivity for the major components of cell membranes, including phosphatidylserine and phosphatidylcholine. To understand how C2 domains show diverse lipid selectivity and how this functional diversity affects their subcellular targeting behaviors, we measured the binding of the C2 domains of group IVa cytosolic phospholipase A 2 (cPLA 2 ) and protein kinase C-␣ (PKC-␣) to vesicles that model cell membranes they are targeted to, and we monitored their subcellular targeting in living cells. The surface plasmon resonance analysis indicates that the PKC-␣ C2 domain strongly prefers the cytoplasmic plasma membrane mimic to the nuclear membrane mimic due to high phosphatidylserine content in the former and that Asn 189 plays a key role in this specificity. In contrast, the cPLA 2 C2 domain has specificity for the nuclear membrane mimic over the cytoplasmic plasma membrane mimic due to high phosphatidylcholine content in the former and aromatic and hydrophobic residues in the calcium binding loops of the cPLA 2 C2 domain are important for its lipid specificity. The subcellular localization of enhanced green fluorescent protein-tagged C2 domains and mutants transfected into HEK293 cells showed that the subcellular localization of the C2 domains is consistent with their lipid specificity and could be tailored by altering their in vitro lipid specificity. The relative cell membrane translocation rate of selected C2 domains was also consistent with their relative affinity for model membranes. Together, these results suggest that biophysical principles that govern the in vitro membrane binding of C2 domains can account for most of their subcellular targeting properties.The agonist-induced subcellular targeting of protein is an important process in cell signaling and regulation. Recently, the membrane targeting of peripheral proteins (e.g. phospholipases, lipid-dependent protein kinases, lipid kinases, and lipid phosphatases) by Ca 2ϩ and lipid mediators, including phosphoinositides, has received much attention as an important event in cell signaling and membrane trafficking. It has been shown that the subcellular targeting of peripheral proteins is driven by a growing number of membrane targeting domains. These domains include protein kinase C (PKC) 1 conserved 1 (C1) domain, PKC conserved 2 (C2) domain, pleckstrin homology (PH) domain, Fab1, YOTB, Vac 1 and EEA1 (FYVE) domain, band four-point-one, ezrin, radixin and moesin (FERM) domain, epsin amino-terminal homology (ENTH) domain, and phox (PX) domains (1-5).The C2 domain has been identified in many cellular proteins involved in signal transduction or membrane trafficking (5-7). A majority of C2 domains bind the membrane in a Ca 2ϩ -dependent manner and thereby play an important role in Ca 2ϩ -dependent membrane targeting of pe...

The regulatory domains of novel protein kinases C (PKC) contain two C1 domains (C1A and C1B), which have been identified as the interaction site for sn-1,2-diacylglycerol (DAG) and phorbol ester, and a C2 domain that may be involved in interaction with lipids and/or proteins. Although recent reports have indicated that C1A and C1B domains of conventional PKCs play different roles in their DAG-mediated membrane binding and activation, the individual roles of C1A and C1B domains in the DAG-mediated activation of novel PKCs have not been fully understood. In this study, we determined the roles of C1A and C1B domains of PKC␦ by means of in vitro lipid binding analyses and cellular protein translocation measurements. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKC␦ have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKC␦ mutants showed that the C1A domain is critical for the DAG-induced membrane binding and activation of PKC␦. The studies also indicated that an anionic residue, Glu 177 , in the C1A domain plays a key role in controlling the DAG accessibility of the conformationally restricted C1A domain in a phosphatidylserine-dependent manner. Cell studies with enhanced green fluorescent protein-tagged PKC␦ and mutants showed that because of its phosphatidylserine specificity PKC␦ preferentially translocated to the plasma membrane under the conditions in which DAG is randomly distributed among intracellular membranes of HEK293 cells. Collectively, these results provide new insight into the differential roles of C1 domains in the DAG-induced membrane activation of PKC␦ and the origin of its specific subcellular localization in response to DAG.

Two novel protein kinases C (PKC), PKC␦ and PKC⑀, have been reported to have opposing functions in some mammalian cells. To understand the basis of their distinct cellular functions and regulation, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKC⑀ and compared it with that of PKC␦. The regulatory domains of novel PKC contain a C2 domain and a tandem repeat of C1 domains (C1A and C1B), which have been identified as the interaction site for DAG and phorbol ester. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKC⑀ have comparably high affinities for DAG and phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKC⑀ mutants showed that both the C1A and C1B domains play a role in the DAG-induced membrane binding and activation of PKC⑀. The C1 domains of PKC⑀ are not conformationally restricted and readily accessible for DAG binding unlike those of PKC␦. Consequently, phosphatidylserinedependent unleashing of C1 domains seen with PKC␦ was not necessary for PKC⑀. Cell studies with fluorescent protein-tagged PKCs showed that, due to the lack of lipid headgroup selectivity, PKC⑀ translocated to both the plasma membrane and the nuclear membrane, whereas PKC␦ migrates specifically to the plasma membrane under the conditions in which DAG is evenly distributed among intracellular membranes of HEK293 cells. Also, PKC⑀ translocated much faster than PKC␦ due to conformational flexibility of its C1 domains. Collectively, these results provide new insight into the differential activation mechanisms of PKC␦ and PKC⑀ based on different structural and functional properties of their C1 domains.

Group IVA cytosolic phospholipase A 2 (cPLA 2 ) has been shown to play a critical role in the agonist-induced release of arachidonic acid. To understand the mechanism by which phosphorylation of Ser 505 and Ser 727 activates cPLA 2 , we systematically analyzed the effects of S505A, S505E, S727A, S727E, S505A/S727A, S505A/S727E, and S505E/S727E mutations on its enzyme activity and membrane affinity. In vitro membrane binding measurements showed that S505A has lower affinity than the wild type or S505E for phosphatidylcholine membranes, which is exclusively due to faster desorption of the membrane-bound S505A. In contrast, neither S727A nor S727E mutation had a significant effect on the phosphatidylcholine vesicle binding affinity of cPLA 2 . The difference in in vitro membrane affinity between wild type (or S505E) and S505A increased with the decrease in Ca 2؉ concentration, reaching >60-fold at 2.5 M Ca 2؉ . When HEK293 cells transfected with cPLA 2 and mutants were stimulated with ionomycin, the wild type and S505E translocated to the perinuclear region and caused the arachidonic acid release at 0.4 M Ca 2؉ , whereas S505A showed no membrane translocation and little activity to release arachidonic acid. Further mutational analysis of hydrophobic residues in the active site rim (Ile 399 , Leu 400 , and Leu 552 ) indicate that a main role of the Ser 505 phosphorylation is to promote membrane penetration of these residues, presumably by inducing a conformational change of the protein. These enhanced hydrophobic interactions allow the sustained membrane interaction of cPLA 2 in response to transient calcium increases. On the basis of these results, we propose a mechanism for cPLA 2 activation by calcium and phosphorylation.

Mammalian secretory phospholipases A 2 (sPLA 2 ) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA 2 mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA 2 acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid, N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA 2 s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA 2 hydrolysis products or cytokines, are necessary for the internalization of sPLA 2 into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA 2 internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA 2 induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA 2 acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis.