The regulatory domains of conventional and novel protein kinases C (PKC) have two C1 domains (C1A and C1B) that have been identified as the interaction site for diacylglycerol (DAG) and phorbol ester. It has been reported that C1A and C1B domains of individual PKC isoforms play different roles in their membrane binding and activation; however, DAG affinity of individual C1 domains has not been quantitatively determined. In this study, we measured the affinity of isolated C1A and C1B domains of two conventional PKCs, PKC␣ and PKC␥, for soluble and membrane-incorporated DAG and phorbol ester by isothermal calorimetry and surface plasmon resonance. The 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. In contrast, the C1A and C1b domains of PKC␥ have comparably high affinities for both DAG and phorbol ester. Consistent with these results, mutational studies of full-length proteins showed that the C1A domain is critical for the DAG-induced activation of PKC␣, whereas both C1A and C1B domains are involved in the DAG-induced activation of PKC␥. Further mutational studies in conjunction with in vitro activity assay and monolayer penetration analysis indicated that, unlike the C1A domain of PKC␣, neither the C1A nor the C1B domain of PKC␥ is conformationally restricted. Cell studies with enhanced green fluorescent protein-tagged PKCs showed that PKC␣ did not translocate to the plasma membrane in response to DAG at a basal intracellular calcium concentration due to the inaccessibility of its C1A domain, whereas PKC␥ rapidly translocated to the plasma membrane under the same conditions. These data suggest that differential activation mechanisms of PKC isoforms are determined by the DAG affinity and conformational flexibility of their C1 domains. Protein kinase C (PKC)1 are a family of serine/threonine kinases that mediate numerous cellular processes (1, 2). All PKCs contain an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Based on structural differences in the regulatory domain, PKCs are generally classified into three groups: conventional PKC (␣, ␤I, ␤II, and ␥ subtypes), novel PKC (␦, ⑀, , and subtypes), and atypical PKC ( and / subtypes). Regulatory domains of both conventional and novel PKCs contain tandem C1 (C1A and C1B) domains and a C2 domain. The C1 domain (ϳ50 residues) is a cysteine-rich compact structure that contains five short ␤ strands, a short helix, and two zinc ions (3-7), whereas the C2 domain (ϳ130 residues) is composed of eight-stranded antiparallel ␤ strands (5, 8 -10) and interconnecting loops some of which are involved in Ca 2ϩ -dependent membrane binding. The C1 domain was first identified as the interaction site for diacylglycerol (DAG) and phorbol ester in PKCs (11), but it was subsequently found in other proteins with diverse functions, including protein kinase D (PKD/PKC), chimaerin, Ras-GRP, DAG kinases, and Raf-1 kinase (4, 6, 7). ...

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.

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The interactions of PI-PLC with nonsubstrate zwitterionic [phosphatidylcholine (PC)] and anionic [phosphatidylmethanol (PMe), phosphatidylserine, phosphatidylglycerol, and phosphatidic acid] interfaces that affect the catalytic activity of PI-PLC have been examined. PI-PLC binding is strongly coupled to vesicle curvature and is tighter at acidic pH for all of the phospholipids examined. PI-PLC binds to small unilamellar vesicles (SUVs) of anionic lipids with much higher affinity (K(d) is 0.01-0.07 microM for a site consisting of n = 100 +/- 25 lipids when analyzed with a Langmuir adsorption isotherm) than to zwitterionic PC SUVs (K(d) is 5-20 microM and n = 8 +/- 3). The binding to PC surfaces is dominated by hydrophobic interactions, while binding to anionic surfaces is dominated by electrostatic interactions. The contributions of specific cationic side chains and hydrophobic groups at the rim of the alpha beta-barrel to zwitterionic and anionic vesicle binding have been assessed with mutagenesis. The results are used to explain how PC activates the enzyme for both phosphotransferase and cyclic phosphodiesterase activities.

Mammalian phospholipases D (PLD), which catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid (PA), have been implicated in various cell signaling and vesicle trafficking processes. Mammalian PLD1 contains two different membrane-targeting domains, pleckstrin homology and Phox homology (PX) domains, but the precise roles of these domains in the membrane binding and activation of PLD1 are still unclear. To elucidate the role of the PX domain in PLD1 activation, we constructed a structural model of the PX domain by homology modeling and measured the membrane binding of this domain and selected mutants by surface plasmon resonance analysis. The PLD1 PX domain was found to have high phosphoinositide specificity, i.e. phosphatidylinositol 3,4,5-trisphosphate (PtdIns-(3,4,5)P 3 ) > > phosphatidylinositol 3-phosphate > phosphatidylinositol 5-phosphate > > other phosphoinositides. The PtdIns(3,4,5)P 3 binding was facilitated by the cationic residues (Lys 119 , Lys 121 , and Arg 179 ) in the putative binding pocket. Consistent with the model structure that suggests the presence of a second lipid-binding pocket, vesicle binding studies indicated that the PLD1 PX domain could also bind with moderate affinity to PA, phosphatidylserine, and other anionic lipids, which were mediated by a cluster of cationic residues in the secondary binding site. Simultaneous occupancy of both binding pockets synergistically increases membrane affinity of the PX domain. Electrostatic potential calculations suggest that a highly positive potential near the secondary binding site may facilitate the initial adsorption of the domain to the anionic membrane, which is followed by the binding of PtdIns(3,4,5)P 3 to its binding pocket. Collectively, our results suggest that the interaction of the PLD1 PX domain with PtdIns(3,4,5)P 3 and/or PA (or phosphatidylserine) may be an important factor in the spatiotemporal regulation and activation of PLD1. Mammalian phospholipase D (PLD)1 catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid (PA) and choline (1, 2). PA may act as a lipid mediator for various proteins involved in cell signaling and vesicle trafficking (3, 4) and may also regulate the physical property of the cellular membranes (5, 6). Two isoforms of mammalian PLDs, PLD1 and PLD2, have been implicated in numerous cellular processes, including vesicle trafficking, cytoskeletal rearrangement, and proliferation (1,3,4,7,8). PLDs are activated in many cell types in response to growth factors, hormones, and neurotransmitters (9). It has been reported that PLD activities are regulated through interactions with a wide variety of molecules, including small GTP-binding proteins, such as ADPribosylation factor (Arf), Rho, Rac, and Cdc42, and protein kinase C isoforms (10 -16).In most mammalian cells, PLD activities have been found associated with the membrane fraction but PLDs show complex membrane localization patterns depending on cell types. While PLD2 is mainly found at the plasma membrane (17), PLD1 shows dynam...

The C2 domain is a Ca 2؉ -dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. To understand the mechanisms by which the C2 domain mediates the membrane targeting of PLC-␦ isoforms, we measured the in vitro membrane binding of the C2 domains of PLC-␦1, -␦3, and -␦4 by surface plasmon resonance and monolayer techniques and their subcellular localization by time-lapse confocal microscopy. The membrane binding of the PLC-␦1-C2 is driven by nonspecific electrostatic interactions between the Ca 2؉ -induced cationic surface of protein and the anionic membrane and specific interactions involving Ca 2؉ , Asn 647 , and phosphatidylserine (PS). The PS selectivity of PLC-␦1-C2 governs its specific Ca 2؉ -dependent subcellular targeting to the plasma membrane. The membrane binding of the PLC-␦3-C2 also involves Ca 2؉ -induced nonspecific electrostatic interactions and PS coordination, and the latter leads to specific subcellular targeting to the plasma membrane. In contrast to PLC-␦1-C2 and PLC-␦3-C2, PLC-␦4-C2 has significant Ca 2؉ -independent membrane affinity and no PS selectivity due to the presence of cationic residues in the Ca 2؉ -binding loops and the substitution of Ser for the Ca 2؉ -coordinating Asp in position 717. Consequently, PLC-␦4-C2 exhibits unique prelocalization to the plasma membrane prior to Ca 2؉ import and non-selective Ca 2؉ -mediated targeting to various cellular membranes, suggesting that PLC-␦4 might have a novel regulatory mechanism. Together, these results establish the C2 domains of PLC-␦ isoforms as Ca 2؉ -dependent membrane targeting domains that have distinct membrane binding properties that control their subcellular localization behaviors.

Activation of the serine/threonine protein kinase Akt/PKB is a multi-step process involving membrane recruitment, phosphorylation, and membrane detachment. To investigate this process in the cellular context, we employed a live-cell fluorescence imaging approach to examine conformational changes of Akt and its membrane association. A fluorescence resonance energy transfer-based reporter of Akt action (ReAktion) reveals a conformational change that is critically dependent on the existence of a phosphorylatable threonine 308 in the activation loop, because mutations to either aspartate or alanine abolished the change. Furthermore, a mutant carrying a phosphorylation mimic at this position showed diminished membrane association, suggesting that this phosphorylation plays an important role of promoting the dissociation of activated Akt from the membrane. In addition, the membrane-associating pleckstrin homology domain was found to associate with the catalytic domain when Thr 308 is phosphorylated, suggesting such an interdomain interaction as a mechanism by which phosphorylation within the catalytic domain can affect membrane association. These studies uncover new regulatory roles of this critical phosphorylation event of Akt for ensuring its proper activation and function.Serine/threonine kinase Akt, also known as protein kinase B, plays important roles in cellular processes such as cell growth, metabolism, proliferation, and survival (1, 2). Its activation can be initiated by various extracellular signals that turn on phosphatidylinositol 3-kinase (PI3K) 2 (3). Following production of phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate at the plasma membrane, Akt is recruited to the membrane from the cytosol, through specific binding of the N-terminal pleckstrin homology (PH) domain to these 3Ј phosphoinositides (4, 5). Two phosphorylation events following translocation of Akt to the plasma membrane have been shown to be critical, leading to the full activation of Akt (6). Phosphatidylinositol-dependent kinase 1 is known to phosphorylate Akt at threonine 308 (Thr 308 in Akt-1) in the activation loop of the catalytic/kinase domain (7,8 Although Akt activation has been elaborately dissected through various PH domain and phosphorylation site mutants (10), some important steps in the activation process are still not clearly understood. Specifically, the mechanism by which active Akt molecules dissociate from the membrane remains to be elucidated (11). Such membrane dissociation is critical for Akt to gain access to various substrates in different subcellular locations by translocating to cytosol and nucleus (4, 12). Our previous studies have shown that nuclear Akt activity accumulates, whereas there are still high levels of 3Ј phosphoinositides present at the plasma membrane, suggesting departure of Akt from the membrane occurs prior to massive degradation of 3Ј phosphoinositides (13).We hypothesize that a conformational change generated within Akt during its activation facilitates its d...