Robinson, Karen Ann; (1994) Protein regulators of kinase C. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Protein Kinase C (PKC) is a family of serine/threonine kinases. The PKC isoforms differ in distribution, substrate and cofactor specificity. This suggests that different isoforms may control different physiological events. The PKC family has a pivotal role in signal transduction, therefore its activity must be tightly regulated. The 14-3-3 family consists of highly conserved protein isoforms, with wide range of putative functions. The most abundant mammalian brain 14-3-3 isoforms inhibited PKC, within a three-fold range. Therefore regions conserved between 14-3-3 isoforms must contain the major PKC interaction sites, and variable 14-3-3 sites must also influence potency of inhibition. Individual PKC isoforms were inhibited by mixed brain 14-3-3 isoforms, with varying potency. This work enabled the primary 14-3-3 interaction site on PKC to be narrowed down to the conserved cysteine-rich region (C1). This was substantiated by evidence that diacylglycerol and phorbol ester, both of which bind the C1 region, interfere with inhibition by 14-3-3. Other proteins share this cysteine-rich region, for example c- raf-1, n-chimaerin, DAG kinase and phospholipase A2(PLA2). However 14-3-3 was unable to modify the activity of PLA2, and 14-3-3 had no PLA2 activity. PKCI (protein kinase C inhibitor), was originally purified from bovine brain. In this study a maize gene similar to bovine PKCI was expressed in E. coli, and purified to homogeneity. Maize and bovine PKCI shared characteristics: similar secondary structure, dimerism, and zinc-binding. However maize PKCI poorly inhibited PKC activity by a maximum of 20[percent]. In the presence of both 14-3-3 and maize PKCI, the effects on PKC were synergistic. Database searches have revealed that PKCI belongs to a new protein family, which share the novel zinc binding site.

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