Protein kinase C (PKC) is a key regulatory enzyme believed to be involved in many cellular processes such as cell growth and differentation, hormone release, platelet activation, and many other events. Phosphorylation of substrate proteins usually requires Ca+2 and phospholipid as well as the second messenger, diacylglycerol (DAG). Biologically active phorbol esters substitute for DAG. Despite abundant current studies on this protein, little is known about the physical entity that generates kinase activity and how various activators exert their influence. We have found that the Ca+2 and the phospholipid requirements of PKC greatly exceed those needed for PKC-membrane binding and that the cofactor (phospholipid, Ca+2, and/or DAG) requirements of PKC activation are dependent on the choice of substrate. In searching for an explanation for this behavior, we observed the interaction of substrates with the phospholipid was a critical aspect of PKC action and all good in vito substrates not only bound to phospholipid but caused aggregation of phospholipid vesicles. These observations illustrate the need to evaluate all of the different interactions of PKC, its substrates and cofactors. This study will investigate the interactions of PKC and other components in various states (e.g. phospholipid vesicles and monolayers) and will determine how the events and their more detailed aspects are important to develoment of kinase activity. Identification of nonaggregated assay systems will be attempted. Metal ion binding properties of PKC will be examined by several techniques including direct binding measurements and fluorescence methods. Selectivity of PKC for membrane structural features (e.g. composition and surface curvature) will be studied to determine the factors involved in PKC-membrane binding and in generating nondissociable membrane-bound PKC. The effect of phosphorylation on protein-membrane assembly will be assessed using two different proteins, histones and myelin basic protein. The effect of Ca+2, DAG or phorbol esters on the conformation and on the dynamics of PKC-membrane assembly and dissociation will be investigated using fluorescence and CD methods. All interactions will be compared to appearance of PKC activity to help define those aspects of the enzyme-substrate- membrane-cofactor complex that are critical to development of kinase activity. Defining the in vitro properties will improve our understanding of how in vivo targets for this enzyme are identified.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038819-04
Application #
3295533
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1987-07-01
Project End
1992-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
4
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Arts and Sciences
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Shen, L; Shah, A M; Dahlback, B et al. (1997) Enhancing the activity of protein C by mutagenesis to improve the membrane-binding site: studies related to proline-10. Biochemistry 36:16025-31
Nelsestuen, G L; Martinez, M B (1997) Steady state enzyme velocities that are independent of [enzyme]: an important behavior in many membrane and particle-bound states. Biochemistry 36:9081-6
Harvey, S B; Nelsestuen, G L (1995) Reaction of nitric oxide and its derivatives with sulfites: a possible role in sulfite toxicity. Biochim Biophys Acta 1267:41-4
Lu, Y; Bazzi, M D; Nelsestuen, G L (1995) Kinetics of annexin VI, calcium, and phospholipid association and dissociation. Biochemistry 34:10777-85
Evans Jr, T C; Nelsestuen, G L (1994) Calcium and membrane-binding properties of monomeric and multimeric annexin II. Biochemistry 33:13231-8
Plager, D A; Nelsestuen, G L (1994) Direct enthalpy measurements of the calcium-dependent interaction of annexins V and VI with phospholipid vesicles. Biochemistry 33:13239-49
Bazzi, M D; Nelsestuen, G L (1992) Interaction of annexin VI with membranes: highly restricted dissipation of clustered phospholipids in membranes containing phosphatidylethanolamine. Biochemistry 31:10406-13
Bazzi, M D; Nelsestuen, G L (1992) Autophosphorylation of protein kinase C may require a high order of protein-phospholipid aggregates. J Biol Chem 267:22891-6
Plager, D A; Nelsestuen, G L (1992) Dissociation of peripheral protein-membrane complexes by high pressure. Protein Sci 1:530-9
Xu, C J; Nelsestuen, G L (1992) Association of alpha-phosphatidylinositol-specific phospholipase C with phospholipid vesicles. Biochim Biophys Acta 1120:49-58

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