Lipid lateral heterogeneity in phosphatidylcholine/phosphatidylserine/diacylglycerol vesicles and its influence on protein kinase C activation

The diacylglycerol (DG)/phorbol ester-dependent translocation of conventional protein kinase C (PKC) isozymes is mediated by the C1 domain, a membrane-targeting module that also selectively binds phosphatidylserine (PS). Using stopped-flow spectroscopy, we dissect the contribution of DG/phorbol esters (C1 ligand) and PS in driving the association and dissociation of the C1 domain from membranes. Specifically, we examine the binding to membranes of the C1B domain of PKC␤ with a substituted Trp (Y123W) whose fluorescence is quenched upon binding to membranes. Binding of this construct (C1B␤-Y123W) to phospholipid vesicles is cooperative with respect to PS content and dependent on C1 ligand, as previously characterized. Stopped-flow analysis reveals that the apparent association rate (k on app ), but not the apparent dissociation rate (k off app ), is highly sensitive to PS content: the 60-fold increase in membrane affinity for vesicles containing no PS compared with 40 mol % PS results primarily from a robust (30-fold) increase in k on app with little effect (2-fold) on k off app . Membrane affinity is also controlled by the content and structure of the C1 ligand. In contrast to PS, these ligands markedly alter k off app with smaller effects on k on app . We also show that the affinity for phorbol ester-containing membranes is 2 orders of magnitude higher than that for DG-containing membranes primarily resulting from differences in k off app . Our data are consistent with a model in which the C1 domain is recruited to the membrane via an initial weak electrostatic interaction with PS, followed by a rapid two-dimensional search for ligand, the binding of which retains the domain at the membrane. Thus, PS drives the initial encounter, and DG/phorbol esters retain the domain on membranes. The decreased effectiveness of DG compared with phorbol esters in retaining the C1 domain on membranes contributes to the molecular dichotomy of the rapid, transient nature of DG-dependent PKC signaling versus the chronic hyperactivity of phorbol ester-activated PKC.The activity of PKC 3 is critical to many signaling pathways, through which PKC elicits a variety of physiological outputs (1, 2). Whereas PKC may be constitutively activated through proteolysis, the principal mechanism for the activation of the conventional (␣, the alternatively spliced ␤⌱ and ␤⌱⌱, ␥) and novel (␦, ⑀, , ) PKC isozymes is via translocation of PKC to the membrane; this translocation is driven by the C1 domain of these PKC isozymes, which binds diacylglycerol (DG) or the functional analogues, phorbol esters (3). The intrinsic membrane affinity of the C1 domain of conventional PKC isozymes is 2 orders of magnitude lower than that of the novel PKC isozymes (4, 5), and effective membrane translocation is only achieved following pre-targeting of PKC to the membrane by engaging its Ca 2ϩ -dependent C2 domain (6). For novel PKC isozymes, the affinity of their C1 domain is sufficiently high that agonist-evoked changes in diacylglycerol are sufficient to recruit n...

Section: Discussionmentioning

confidence: 75%

Kinetic Analysis of the Interaction of the C1 Domain of Protein Kinase C with Lipid Membranes by Stopped-flow Spectroscopy

Dries

Newton

2008

“…Recent experiments in a DMPC/DMPS/DAG system revealed a peak in PKC activity at DAG mol fractions that corresponded to a mixture of compositionally distinct lipid domains as determined by differential scanning calorimetry (32,33). Potential explanations for the decrease in activity at high mol % DAG included (a) some requirement for interface regions between domains that would be maximal under maximal domain coexistence conditions; (b) a possible dilution of PKC multimers or PKC-substrate aggregates as an optimal domain is increased; and (c) the decrease in mol % PS that would occur as DAG was increased at the expense of PC and PS in those studies.…”

Section: Resultsmentioning

confidence: 99%

“…32 P incorporation into PKC was determined by autoradiography after polyacrylamide gel electrophoresis using XK-1 film. Several exposures were obtained to ensure linearity of detection, and autophosphorylation was quantitated by densitometry.…”

Section: Methodsmentioning

confidence: 99%

“…These include short chain PCs (21) and short chain PSs (27), lysophospholipids (28), steroids such as tamoxifen (29) and bile acids (30), and free fatty acids (31). In saturated phospholipid systems, a maximum is observed as a function of DAG (32,33), suggesting several hypotheses having to do with the coexistence of compositionally distinct domains and/or interface regions in addition to alterations in mol % PS. Goldberg and Zidovetski (31) observed similar maxima as a function of DAG in unsaturated systems and noted a correlation between increasing activity and increasing frustration of the bilayer, but decreasing activity as bilayers were disrupted to form a HexII phase.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Contributions to Maxima in Protein Kinase C Activation

Sando¹,

Chertihin²,

Owens

et al. 1998

In many lipid systems, the activity of protein kinase C (PKC) exhibits a peak followed by a decline as the mol % of one component is increased. In these systems, an increase in one lipid component is always at the expense of another or accompanied by a change in total lipid concentration. Here we report that in saturated phosphatidylserine (PS)/phosphatidylcholine (PC)/diacylglycerol (DAG) mixtures, increasing PS or DAG at the expense of PC revealed an optimal mol % PS, dependent on mol % DAG, with higher mol % PS diminishing activity. The decrease at high mol % PS is probably not attributable simply to more gel-phase lipid due to the higher melting temperature of saturated PS versus PC because a similar peak in activity occurred in unsaturated lipid systems. Increasing the total lipid concentration at suboptimal mol % PS provided the same activity as higher mol % PS at lower total lipid concentration. However, at optimal mol % PS, activity increased and then decreased as a function of total lipid concentration. PKC autophosphorylation also exhibited an optimum as a function of mol % PS, and increasing the PKC concentration increased the mol % PS at which activity decreased, both for autophosphorylation and for heterologous phosphorylation. Formation of two-dimensional crystals of PKC on lipid monolayers also exhibited a peak as a function of mol % PS, and the unit cell size of the crystals formed shifts from 50 ؋ 50 Å at low mol % PS to 75 ؋ 75 Å at higher PS. Collectively, these data suggest the existence of optimal lipid compositions for PKC activation, with increased quantity of these domains serving to dilute out enzyme-substrate aggregates and/or enzyme-enzyme aggregates on the lipid surface.Protein kinase C (PKC) 1 (1), which constitutes a family of structurally related kinases, is defined by the phospholipid dependence of its activity, with all isozymes activated by acidic phospholipids, preferably phosphatidylserine (PS), and all but PKC and PKC/ further stimulated by diacylglycerols (DAG) or phorbol esters. The originally identified ␣, ␤, and ␥ family members also require calcium binding to the C2 domain, which is distinct in these isozymes (for reviews, see Refs. 2-4). Although the role of some lipid components is becoming better defined, variable results in different systems leave many questions unanswered.Structural analyses of DAG and phorbol ester analogues (5-7) and analysis of PKC deletions and mutations argue for specific phorbol ester and DAG binding to the Cys-rich, zinccontaining C1 domain (8 -11). X-ray fluorescence (12), NMR (13), and x-ray crystallography (14) have revealed the structure of this domain. Binding of phorbol ester does not alter the conformation, but helps to present a hydrophobic surface on the domain that could facilitate insertion into the membrane to a depth of 7-8 Å (14). The two versions of the C1 domain, C1A and C1B, present in most PKC isozymes have structural differences (reviewed in Ref. 15) that may account for greatly differing affinities of fluorescent phorbol e...

“…In this regard, it is noteworthy that DAG has been shown to induce local PS-rich membrane domains (46). The preferential binding of conventional PKCs to such domains would trigger the membrane penetration and DAG binding.…”

Section: Discussionmentioning

confidence: 97%

Interplay of C1 and C2 Domains of Protein Kinase C-α in Its Membrane Binding and Activation

Medkova

Cho

1999

157

184

The regulatory domain of conventional protein kinase C (PKC) contains two membrane-targeting modules, the C2 domain that is responsible for Ca 2؉ -dependent membrane binding of protein, and the C1 domain composed of two cysteine-rich zinc fingers (C1a and C1b) that bind diacylglycerols and phorbol esters. To understand the individual roles and the interplay of the C1 and C2 domains in the membrane binding and activation of PKC, we functionally expressed isolated C1 and C2 domains of PKC-␣ and measured their vesicle binding and monolayer penetration. Results indicate that the C2 domain of PKC-␣ is responsible for the initial Ca 2؉ -and phosphatidylserine-dependent electrostatic membrane binding of PKC-␣, whereas the C1 domain is involved in subsequent membrane penetration and diacylglycerol binding, which eventually lead to enzyme activation. To determine the roles of individual zinc fingers in the C1 domain, we also mutated hydrophobic residues in the C1a (Trp 58 and Protein kinases C (PKCs)1 are a family of serine/threonine kinases that transduce the myriad of signals activating cellular functions and proliferation (1, 2). More than 10 members of the PKC family have been identified by molecular cloning. 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). The regulatory domain of conventional PKCs is composed of two conserved membrane-targeting modules, C1 and C2 domains, as well as a pseudosubstrate region and variable regions. Conventional PKCs are activated by the Ca 2ϩ -dependent translocation of proteins to the membrane containing phosphatidylserine (PS) and diacylglycerol (DAG). Structural (3) and mutational (4, 5) studies have shown that the C2 domain of conventional PKC is responsible for the Ca 2ϩ -dependent translocation of the protein to membranes. It has also been shown that the C1 domain, which is composed of a tandem repeat of cysteine-rich zinc-finger domains (C1a and C1b), is involved in binding of PKC to DAG and its structural analogs, phorbol esters (6 -8). However, the temporal and spatial sequences of membrane targeting and activation of PKC have not been fully elucidated. Furthermore, the interplay of the C1 and C2 domains in these complex processes is poorly understood. Finally, the roles of individual zinc finger domains in the DAGdependent membrane binding and activation of PKC are not well defined. To address these questions, we functionally expressed the isolated C1 and C2 domains of PKC-␣ and measured their vesicle binding and monolayer penetration. We also mutated C1a and C1b domain residues of the native PKC-␣ and measured the effects of mutations on vesicle binding, enzyme activity, and monolayer penetration. Results indicate that the C2 domain of PKC-␣ is responsible for its initial Ca 2ϩ -and PS-dependent membrane bind...