Protein kinase C (PKC) is translocated to a phospholipid bilayer by calcium. Once at the membrane protein kinase C undergoes a conformational change which results in the removal of the pseudosubstrate domain from the active site. The enzyme then phosphorylates Ser/Thr residues on positively charged substrates. Certain substrates, however, can undergo cofactor independent phosphorylation by producing a conformational change in the enzyme in the absence of phospholipid and calcium. Studying the conformational change in PKC by physical techniques is difficult to perform with a phospholipid bilayer present. To study the conformational change in PKC in the absence of a membrane, the interactions between an Arginine-rich peptide (ARP), which underwent cofactor independent phosphorylation, and PKC was investigated. The Kₘ and kcₐₜ of the enzyme for ARP, in the absence of cofactors, was around 10 μM and 0.38 s⁻¹, respectively. The Kₘ did not significantly change upon the addition of phosphoipid and calcium. However, the kcₐₜ increased 2-3 fold in the presence of phospholipid and calcium. In the absence of phospholipid and calcium, ARP induced the exposure of hydrophobic site(s) on the enzyme. Additionally, ARP was able to promote the translocation of PKC to the membrane in the absence of calcium. PKC translocated to the membrane by ARP displayed the same susceptibility as the calcium membrane bound enzyme to limited proteolytic cleavage. Therefore, both ARP and calcium induce a similar membrane bound conformation in PKC. Additionally, the binding of ARP to PKC seems to occur through at least one high affinity site apart for the active site. These results demonstrate new insight into cofactor independent phosphorylation by PKC as well as illustrate a novel mechanism by which a substrate can promote the translocation of PKC in the absence of calcium. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22790 |
| Date | 09 1900 |
| Creators | Bruins, Robert |
| Contributors | Epand, Richard, Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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