Protein kinase C is specifically activated by binding two membrane lipids: the second messenger, diacylglycerol, and the amino phospholipid, phosphatidylserine. This binding provides the energy to release an autoinhibitory pseudosubstrate from the active site. Interaction with these lipids recruits the enzyme to the membrane by engaging two membrane-targeting modules: the C1 domain (present as a tandem repeat in most protein kinase Cs) and the C2 domain. Here we dissect the contribution of each domain in recruiting protein kinase C betaII to membranes. Binding analyses of recombinant domains reveal that the C2 domain binds anionic lipids in a Ca(2+)-dependent, but diacylglycerol-independent, manner, with little selectivity for phospholipid headgroup beyond the requirement for negative charge. The C1B domain binds membranes in a diacylglycerol/phorbol ester-dependent, but Ca(2+)-independent manner. Like the C2 domain, the C1B domain preferentially binds anionic lipids. However, in striking contrast to the C2 domain, the C1B domain binds phosphatidylserine with an order of magnitude higher affinity than other anionic lipids. This preference for phosphatidylserine is, like that of the full-length protein, stereoselective for sn-1, 2-phosphatidyl-L-serine. Quantitative analysis of binding constants of individual domains and that of full-length protein reveals that the full-length protein binds membranes with lower affinity than expected based on the binding affinity of isolated domains. In addition to entropic and steric considerations, the difference in binding energy may reflect the energy required to expel the pseudosubstrate from the substrate binding cavity. This study establishes that each module is an independent membrane-targeting module with each, independently of the other, containing determinants for membrane recognition. The presence of each of these modules, separately, in a number of other signaling proteins epitomizes the use of these modules as discreet membrane targets.

Protein kinase C (PKC) family members are allosterically activated following membrane recruitment by specific membranetargeting modules. Conventional PKC isozymes are recruited to membranes by two such modules: a C1 domain, which binds diacylglycerol (DAG), and a C2 domain, which is a Ca 2؉ -triggered phospholipid-binding module. In contrast, novel PKC isozymes respond only to DAG, despite the presence of a C2 domain. Here, we address the molecular mechanism of membrane recruitment of the novel isozyme PKC␦. We show that PKC␦ and a conventional isozyme, PKC␤II, bind membranes with comparable affinities. However, dissection of the contribution of individual domains to this binding revealed that, although the C2 domain is a major determinant in driving the interaction of PKC␤II with membranes, the C2 domain of PKC␦ does not bind membranes. Instead, the C1B domain is the determinant that drives the interaction of PKC␦ with membranes. The C2 domain also does not play any detectable role in the activity or subcellular location of PKC␦ in cells; in vivo imaging studies revealed that deletion of the C2 domain does not affect the stimulus-dependent translocation or activity of PKC␦. Thus, the increased affinity of the C1 domain of PKC␦ allows this isozyme to respond to DAG alone, whereas conventional PKC isozymes require the coordinated action of Ca 2؉ binding to the C2 domain and DAG binding to the C1 domain for activation.Many signaling proteins translocate from the cytosol to the membrane to become activated or to reach their substrates. Reversible binding to the membrane is mediated by modules such as the C1, C2, pleckstrin homology, and FYVE domains, with each recognizing specific lipid determinants or membrane properties. The protein kinase C (PKC) 3 family is a classic example of an enzyme whose function is regulated by membrane translocation. For this family of signaling proteins, the C1 and C2 domains direct the enzyme to the membrane following generation of diacylglycerol (DAG) and, for some isoforms, CaThe C1 domain is a 5-kDa globular domain composed primarily of two ␤-sheets that are pulled apart to expose a binding pocket for DAG and phorbol. Found in a number of other proteins in addition to PKC (notably, protein kinase D, DAG kinase, and Raf), this domain is an effective DAG sensor when expressed alone (reviewed in Refs. 2 and 3). Moreover, the C1 domain selectively recognizes phosphatidylserine (PS) (4, 5). The C2 domain is another small (16 kDa) globular domain that is primarily composed of ␤-strands; in this case, three loops in the ␤-strands at one end of the domain form a Ca 2ϩ -binding site. Conventional C2 domains such as those found in phospholipase C, phospholipase A 2 , phosphatidylinositol 3-kinase, synaptotagmin, and Munc13 bind Ca 2ϩ in this pocket, and the binding drives the membrane interaction (reviewed in Refs. 6 and 7). Novel C2 domains such as that found in PTEN do not bind Ca 2ϩ , yet can still bind phospholipids (8). The coordinated use of a C1 and C2 domain allows sensitivity in the...

BackgroundThe uptake of particles by actin-powered invagination of the plasma membrane is common to protozoa and to phagocytes involved in the immune response of higher organisms. The question addressed here is how a phagocyte may use geometric cues to optimize force generation for the uptake of a particle. We survey mechanisms that enable a phagocyte to remodel actin organization in response to particles of complex shape.ResultsUsing particles that consist of two lobes separated by a neck, we found that Dictyostelium cells transmit signals concerning the curvature of a surface to the actin system underlying the plasma membrane. Force applied to a concave region can divide a particle in two, allowing engulfment of the portion first encountered. The phagosome membrane that is bent around the concave region is marked by a protein containing an inverse Bin-Amphiphysin-Rvs (I-BAR) domain in combination with an Src homology (SH3) domain, similar to mammalian insulin receptor tyrosine kinase substrate p53. Regulatory proteins enable the phagocyte to switch activities within seconds in response to particle shape. Ras, an inducer of actin polymerization, is activated along the cup surface. Coronin, which limits the lifetime of actin structures, is reversibly recruited to the cup, reflecting a program of actin depolymerization. The various forms of myosin-I are candidate motor proteins for force generation in particle uptake, whereas myosin-II is engaged only in retracting a phagocytic cup after a switch to particle release. Thus, the constriction of a phagocytic cup differs from the contraction of a cleavage furrow in mitosis.ConclusionsPhagocytes scan a particle surface for convex and concave regions. By modulating the spatiotemporal pattern of actin organization, they are capable of switching between different modes of interaction with a particle, either arresting at a concave region and applying force in an attempt to sever the particle there, or extending the cup along the particle surface to identify the very end of the object to be ingested. Our data illustrate the flexibility of regulatory mechanisms that are at the phagocyte's disposal in exploring an environment of irregular geometry.

The hallmark for protein kinase C activation is its "translocation" to membranes following generation of lipid second messengers. This translocation is mediated by the C1 and C2 domains, two membrane-targeting modules, whose engagement on membranes provides the energy for an activating conformational change in which an autoinhibitory pseudosubstrate sequence is released from the active site. Novel and conventional protein kinase C isozymes contain a tandem repeat of C1 domains, the C1A and C1B, which each contain a binding pocket for phorbol esters/diacylglycerol. This study addresses the contribution of the C1A and C1B domains in the regulation of protein kinase C's membrane interaction using bisfunctional (dimeric) phorbol myristate acetate (PMA) molecules. We show that dimeric bisphorbols are an order of magnitude more effective at recruiting full-length PKC betaII to membranes compared with monomeric PMA and that the effectiveness of the interaction depends on the nature and length of the cross-link between the PMA moieties. Most effective were dimeric phorbol 12-acetate 13-esters linked at the 13 position with a 14 carbon spacer. The increased potency of dimeric phorbol esters is reduced if either the C1A or C1B domains are mutated so that they are unable to bind PMA, if one moiety of the dimer contains a nonfunctional phorbol, or if the binding to the isolated C1B domain is measured. Thus, the increased potency of the dimeric phorbol esters results primarily from their ability to engage, to a limited extent, both C1 modules on the same molecule. Although dimeric phorbols were more potent than monomeric phorbol esters in recruiting protein kinase C to membranes, the magnitude of the increase was still several orders of magnitude lower than what would be predicted on the basis of the reduction in dimensionality that occurs when the first C1 domain is engaged on the membrane. Thus, engaging both domains can be forced but is highly unfavored. In summary, our data reveal that both C1 domains are oriented for potential membrane interaction but only one C1 domain binds ligand in a physiological context.

Alterations in mitochondrial function contribute to diabetic cardiomyopathy. We have previously shown that heart mitochondrial proteins are hyperacetylated in OVE26 mice, a transgenic model of type 1 diabetes. However, the universality of this modification and its functional consequences are not well established. In this study, we demonstrate that Akita type 1 diabetic mice exhibit hyperacetylation. Functionally, isolated Akita heart mitochondria have significantly impaired maximal (state 3) respiration with physiological pyruvate (0.1 mm) but not with 1.0 mm pyruvate. In contrast, pyruvate dehydrogenase activity is significantly decreased regardless of the pyruvate concentration. We found that there is a 70% decrease in the rate of pyruvate transport in Akita heart mitochondria but no decrease in the mitochondrial pyruvate carriers 1 and 2 (MPC1 and MPC2). The potential role of hyperacetylation in mediating this impaired pyruvate uptake was examined. The treatment of control mitochondria with the acetylating agent acetic anhydride inhibits pyruvate uptake and pyruvate-supported respiration in a similar manner to the pyruvate transport inhibitor α-cyano-4-hydroxycinnamate. A mass spectrometry selective reactive monitoring assay was developed and used to determine that acetylation of lysines 19 and 26 of MPC2 is enhanced in Akita heart mitochondria. Expression of a double acetylation mimic of MPC2 (K19Q/K26Q) in H9c2 cells was sufficient to decrease the maximal cellular oxygen consumption rate. This study supports the conclusion that deficient pyruvate transport activity, mediated in part by acetylation of MPC2, is a contributor to metabolic inflexibility in the diabetic heart.