Lipids and

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Lipids and

    Lipids and Protein Kinase C

Introduction to PKC The protein Kinase C (PKC) family transduces cell signaling

    pathways that hydrolyse lipids (Newton, 1995). In response to diacylglycerol (DAG) in the membrane, PKC phosphorylates specific substrates. Although activated in a wide range of cellular processes, a primary role of PKC is receptor desensitization (Newton 1995).

    PKC consists of C-terminal catalytic domains (45 kDa) and N-terminal regulatory domains (20-40kDa). The C3 and C4 (Figure One) catalytic domain bind ATP and the substrate. They function in a similar fashion to Protein Kinase A, although not as specifically. The C2 domain interacts with phosphoserine (PS) lipids in the bilayer. This reversible binding keeps PKC associated with the cytosolic side of the membrane. The C1 domain interacts with DAG in the bilayer. C1 has a beta-sheet form, and acts by removing the pseudo-substrate from PKC’s

    substrate binding domain.

    PKC exists in a wide range of isozymes and in vivo both PKC and substrates are localized. PKC is regulated by phosphorylation (PDK-1 Newton, 2002), PS, DAG as well as non-specific interactions membrane lipids.

    PKC can be activated in vitro via DAG or ester analogues that can’t be decomposed by the cell. PKC can be over-expressed and GFP labeled. Binding of PKC to lipid can be assayed by mixing vesicles with PKC and separating the resultant solution by centrifugation. The fraction of protein in each phase is measured by UV absorption spectroscopy. PKC activity is 32monitored by P assay of phosphorylation.

    PKC and lipids - Although PKC can operate in solution, activity dramatically increases when bound to the membrane. Lipid head-groups, lipid chains, bilayer membrane potential, salt concentration and anesthesia (Slater et. al., 1993) all alter PKC activity. Considerable debate continues about the role of the membrane. Alexandra Newton (UC San Diego) states, “Lipid Structure and Not Membrane Structure is the Major Determinant in the Regulation of Protein Kinase C by Phosphatidylserine” (Johnson et al, 1998). In contrast, Raphael Zidovetzki believes “the specific membrane parameter associated with PKC activation is the increased tendency to form non-bilayer lipid structure, promoted by the addition of the lipids with high intrinsic curvature.” (Huang et al., 1999)

    Reasonable experimental evidence exists for both views. Under physiological conditions, PKC certainly responds to the chirality of lipids within the bilayer. Figure Two illustrates the clear effect of reversing PS handedness. However, there is also considerable evidence for the role of the membrane. Zidovetzki added fatty acids and DAG which increases spontaneous curvature of the bilayer and as shown in Figure Three, markedly altered PKC activity. Similarly, ceramide and DAG act together as shown in Figure Four (Huang et al, 1999). Work by Slater et al. examining the effect of unsaturation of PC and PE lipids on PKC activity (Figure 5) showed a clear correlation between curvature and PKC (Slater et al., 1994).

    Because PKC has a plethora of interactions it is difficult to establish that membrane curvature is a significant modulator of it’s activity. Authors of a number of studies have highlighted other membrane properties.

    One curious fact is that lyso-PCs and lyso-PAs both activate PKC in certain membrane mixtures (Sando et al., 1996). This effect certainly involves co-existing gel and liquid phases. While DMPC/DMPS membranes were strongly activated, DOPC/DOPS bilayers were

    unaffected by lyso-PCs. Further study by Sando’s group (Hinderlitter et al., 1997) revealed (for the DMPC/DMPS system) the peak DAG activation of PKC occurred near the mid-point of gel-lc co-existence. The suggestion that PKC is activated by gel-liquid phase boundaries seems plausible in this system, but does not apply to the fluid bilayer.

    In recent years, Richard Epand has argued that PKC activation is regulated by the energy of hydrophobic access into the bilayer rather than by strain or frustration within the bilayer. This is an important distinction, but a difficult one to clarify as spontaneous curvature is coupled to the energetics of insertion. The simple fact that PKC activates in Triton micelles with PS:DAG (Y.A. Hannun, C.R. Loomis, R.M. Bell. J Biol. Chem. 260 (1985) 10039-10043) confirms that strain energy is not essential for enzymatic activation.

    To separate bilayer strain and burial energy, Epand and coworkers studied PKC in lamellar, cubic and hexagonal phases (Giorgione et al, 1998). Measuring both binding and enzymatic activity, they calculated a “specific activation” of PKC. For the cubic phase (Monoolein:PS or DEPE:alamethicin) this “specific activation” was higher than the corresponding lamellar case (PC:PS). Cubic phases should have lower “strain energy” suggesting PKC does not couple to spontaneous curvature. Proper equilibration of samples, difficulties in measuring “specific activation” and the need to compare markedly different lipid systems are all concerns raised by this study.

    In a second study (Giorgione et al., 1998b) Epand examined the effects of DPLPE, DOPE and DVPE on PKC activation. Although DOPE had the strongest effect and has the greatest non-lamellar tendency, Epand examined the quenching of DTMAC by 5-doxyl-PC as a probe of the energy of reaching the hydrophobic core. Quenching also correlated well with PKC activity. In a more recent study, Drobnies et al. (2002) examined the effects of PAPC and diOH-PAPC in a PC:PS host membrane. Although PAPC has the greater lamellar tendency, it induced marginally higher activation of PKC. In contrast, diOH-PAPC reduces access to the hydrophic core (assayed by DMTPC quenching) and had a reduced activation of PKC. This confirms the role of the insertion energy into the hydrophobic core.

    These studies confirm the complexity of PKC. PS and DAG have a specific interaction with PKC although other lipids can fulfill their roles. Access to the hydrophobic core is significant and would explain why both spontaneous curvature and the co-existence of gel-liquid states activate PKC under appropriate conditions. Spontaneous curvature certainly plays some role, and may indeed by the “specific membrane parameter” (Zidovetzki) under physiological conditions.

Zidovetzki’s Lipid Mixtures

Zidovetzki’s experimental system is.

    Lipids - Bovine Liver PC, DPPC and DPPS (3:1:1) mixture added to

     DAG of some kind (up to 25%) + Fatty Acid or Ceramide (up to 25%)

    Solution for Assays - 20mM MOPS (pH 7.4) + 5mM MgCl2 + 40 microM

    CaCl2 + protein stuff.

    Solutions for NMR - 40mM MOPS + 65mM MgCl2

    These mixtures have been studied primarily via NMR. The known points on the phase diagram for the fatty acids are shown in Figure Six. At 30C, phase coexistence is considerable. With the exception of palmitic acid, though, the gel phase is rarely present.

    Studies of Ceramide:6 are less extensive and consistently show a gel-like phase. A bilayer with 15% DOG and 20% ceramide does induce an isotropic phase at 60C. DSC scans of natural ceramide added to DEPE s confirm cermide lowers the lamellar to hexagonal phase transition temperature and may well induce a cubic phase (Veiga, et al. 1999).

These lipid mixtures are poorly understood.

Proposed Work

    Two major difficulties to studying these mixtures are phase coexistence and sample equilibration. Although the samples do have charged lipids, 20% PS with a reasonable amount of divalent cations could well be manageable. Nola Fuller and Peter Rand believe they are achieving reliable results with a 1:4 PS:PE mixture. Phase coexistence could be an even greater problem.

    It is worth seeing how ordered these lipid mixtures can be made. Good representative samples are,

    1. A sample with 25% DHA/AA and 25% DAG would require sample equilibration

    but hasn’t much phase coexistence. If the sample is poorly ordered, another without

    PS or divalent counterions could be made to check the charged lipids are the


    2. A sample with 15% DOG and 20% C6-ceramide would address the potential of

    these mixtures. There is an isotropic phase at 60C that may be well ordered. Again,

    an uncharged mixture could be tested if necessary.

    If these samples aren’t too difficult to handle then much of phase space should be accessible. Should they not order or equilibrate well then the project is probably too difficult.


    Raphael Zidovetzki and colleagues have established a direct correlation between membrane composition and PKC activity. As spontaneous curvature is likely to be a significant factor, it is worth seeing if X-ray diffraction can determine the spontaneous curvature of these mixtures.


    Drobnies, Adrienne E., Sarah M.A. Davies, Ruud Kraayenhof, Raquel F. Epand, Richard M. Epand, Rosemary B. Cornell. 2002. CTP:phosphocholine cytidylyltransferase and protein kinase C recognize different physical features of membranes : differential responses to an oxidized phosphatidylcholine. Biochimica et Biophysica Acta 1564 : 82-90.

    Epand, Richard M.1998. Lipid polymorphism and protein-lipid interactions. Biochimica et Biophysica Acta 1376 : 353-368.

    Giorgione, Jennifer R., Zhi Huang and Richard M. Epand. 1998. Incrased Activation of Protein Kinase C with Cubic Phase Lipid Compared to Liposomes. Biochemistry 37 : 2384-2392.

    Giorgione, Jennifer, Ruud Kraayenhof and Richard Epand. 1998b. Interfacial Membrane Properties Modulate Protein Kinase C Activation : Role of the position of Acyl Chain Unsaturation. Biochemistry 37: 10965.

    Goldberg, Edward M., David S. Lester, Dan B. Borchardt, Raphael Zidovetzki. 1994. Effects of Diacylglycerol and Ca++ on Structure of Phosphatidylcholine /Phosphatidylserine Bilayers. Biophysical Journal 66: 382-393.

    Goldberg, Edward M. and Raphael Zidovetzki. 1997. Effects of Dipalmitoylglycerol and Fatty Acids on Membrane Structure and Protein Kinase C Activity. Biophysical Journal 73: 2603-2614.

    Goldberg, Edward M. and Raphael Zidovetzki. 1998. Synergistic Effects of Diacylglycerols and Fatty Acids on Membrane Structure and Protein Kinase C Activity. Biochemistry 37: 5623-5632.

    Hinderliter, Anne K., Andrew R.G. Dibble, Rodney L. Biltonen and Julianne J. Sando. 1997. Activation of Protein Kinase C by Coexisting Diacylglycerol-Enriched and Diacylglycerol-Poor Lipid Domains. Biochemistry 36: 6141-4148.

    Hsueh, Ya-Wei, Ralph Giles, Neil Kitson and Jenifer Thewalt. 2002. The Effect of Ceramide on Phosphatidylcholine Membranes : A Deuterium NMR Study. Biophysical Journal 82: 3089-3095.

    Huang, Hsiao-wen, Edward M. Goldberg, Raphael Zidovetzki. 1998. Ceramides perturb the structure of phosphatidylcholine bilayers and modulate the activity of phospholipase A2. European Biophysical Journal 27: 361-366.

    Huang, Hsaio-Wen, Edward M. Goldberg, Raphael Zidovetzki. 1999. Ceramides Modulate Protein Kinase C Activity and Perturb the Structure of Phosphatidylcholine/ Phosphatidylserine Bilayers. Biophysical Journal 77 : 1489-1497.

    Johnson, Joanne E., Michael L. Zimmerman, David L. Daleke, Alexandra C. Newton. 1998. Lipid Structure and Not Membrane Structure Is the Major Determinant in the Regulation of Protein Kinase C by Phosphatidylserine. Biochemistry 37: 12020-12025.

    Massey, John B. 2001. Interaction of ceramides with phosphatidylcholine, sphinogmyelin and sphingomyelin/cholesterol bilayers. Biochimica et Biophysica Acta 1510 : 167-184.

    Newton, Alexandra C. 1995. Protein Kinase C : Structure, Function and Regulation. The Journal of Biological Chemistry 270(48) : 28495-29498.

    Newton, Alexandra C. 2002. Analyzing Protein Kinase C Activation. Methods in Enzymology 345:499-506.

    Sando, Julianne J. and Olga I. Chertihin. 1996. Activation of protein kinase C by lysophosphatidic acid : dependence on composition of phospholipid vesicles. Biochemical Journal 317: 583-588.

    Slater, Simon J., Kingsley, J.A. Cox, Joseph V. Lombardi, Cojen Ho, Mary Beth Kelly, Emanuel Rubin and Christopher D. Stubbs. 1993. Inhibition of protein kinase C by alcohols and anaesthetics. Nature 364:82-84

    Slater, Simon J., Mary Beth Kelly, Frank J. Taddeo, Cojen Ho, Emanuel Rubin and Christopher D. Stubbs. 1994. The Modulation of Protein Kinase C Activity by Membrane Lipid Bilayer Structure. Journal of Biological Chemistry 269:4866-4871.

    Szule, Joseh A., Nola L. Fuller and R. Peter Rand. 2002. The Effects of Acyl Chain Length and Saturation of Diacylglycerols and Phosphatidylcholines on Membrane Monolayer Curvature. Biophysical Journal 83:977-984.

    Veiga, M. pilar, Jose Luis R. Arrondo, Felix M Goni, Alicia Alonso. 1999. Ceramides in Phospholipid Membranes : Effects on Bilayer Stability and Transition to Nonlamellar Phases. Biophysical Journal 76: 342-350.

    Zidovetzki, Raphael. 1997. Membrane Properties and the Activation of Protein Kinase C and Phospholipase A2. Current Topics in Membranes 44: 255-283.

Figure One (Newton, 2002) PKC Structure and Regulation.

Figure Two Chiral Sensitivity of PKC (Johnson et al, 1998)

Figure Three DAG + Fatty Acid activation of PKC (Goldberg and Zidovetzki, 1998)

Figure Four DAG + Ceramide Activate PKC (Huang et al, 1999)

Figure Five PKC Activation by PC and PE Chain Variation (Slater, 1994)

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