Bruins R H, Epand R M
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Arch Biochem Biophys. 1995 Dec 20;324(2):216-22. doi: 10.1006/abbi.1995.0033.
The protein kinase C-alpha (PKC-alpha)-catalyzed phosphorylation of the peptide [Arg]4-Tyr-Gly-Ser-[Arg]5-Tyr is independent of Ca2+ and phospholipid. The binding of this peptide to PKC-alpha induces a conformational change in the enzyme that results in the exposure of hydrophobic groups that subsequently insert into a membrane. Induction of a conformational change in the enzyme by this peptide is demonstrated by susceptibility to trypsin cleavage. Additionally, exposure of hydrophobic sites on the enzyme is shown by the binding of the fluorescent probes PRODAN and bis-ANS and by the partitioning of the enzyme into a Triton X-114-enriched phase. In the presence of a phospholipid bilayer containing phosphatidylserine, this peptide promotes the translocation of PKC-alpha to the membrane in the absence of Ca2+ as observed by increased resonance energy transfer between Trp on the enzyme and dansyl-groups attached to the lipid, as well as by changes in the intrinsic tryptophan fluorescence of the enzyme. Also, once bound to the membrane the peptide.PKC-alpha complex undergoes further conformational change which is evident by an increased sensitivity to trypsin cleavage at the hinge region. These results demonstrate that substrate binding can also induce translocation of PKC to the membrane and suggest that the removal of the pseudosubstrate domain is coupled to a conformational change in the enzyme that results in the exposure of hydrophobic groups.
蛋白激酶C-α（PKC-α）催化的肽段[精氨酸]4-酪氨酸-甘氨酸-丝氨酸-[精氨酸]5-酪氨酸的磷酸化不依赖于Ca2+和磷脂。该肽段与PKC-α的结合会诱导酶发生构象变化，导致疏水基团暴露，随后插入膜中。该肽段对酶构象变化的诱导通过对胰蛋白酶切割的敏感性得以证明。此外，荧光探针PRODAN和双-ANS的结合以及酶在富含Triton X-114的相中分配，表明酶上疏水位点的暴露。在含有磷脂酰丝氨酸的磷脂双层存在下，如通过酶上色氨酸与连接在脂质上的丹磺酰基团之间共振能量转移的增加以及酶内在色氨酸荧光的变化所观察到的，该肽段在没有Ca2+的情况下促进PKC-α向膜的转位。而且，一旦与膜结合，肽-PKC-α复合物会发生进一步的构象变化，这在铰链区对胰蛋白酶切割的敏感性增加中很明显。这些结果表明底物结合也可以诱导PKC向膜的转位，并表明假底物结构域的去除与酶的构象变化相关联，导致疏水基团的暴露。
