Abstract
Peptides Derived from the C2 Domain of Protein Kinase Cϵ (ϵPKC) Modulate ϵPKC Activity and Identify Potential Protein-Protein Interaction Surfaces
Highlights
The protein kinase C (PKC)3 family of serine/threonine protein kinases is involved in normal cell functions such as apoptosis [3, 4], cell proliferation [5,6,7], and secretion [8], as well as in disease states such as ischemic heart disease (9 –12) and stroke [13, 14]
The two peptides, 11 and 13, that induced myristoylated alanine-rich C kinase substrate (MARCKS) phos- selectively modulate ⑀PKC activity; one peptide interferes with phorylation in both wild-type and ⑀KO cells (Fig. 3, A versus C) ⑀PKC interaction with its anchoring protein, ⑀RACK (⑀V1–2 pepand are not ⑀PKC-selective were not cardioprotective, tide) [22], and the other interferes with PKC autoinhibitory possibly because in addition to ⑀PKC they activate the opposing intramolecular interactions (⑀RACK peptide) [12]
Because these isozyme ␦PKC [11]
Summary
Peptide Synthesis—Peptides were synthesized and conjugated to TAT carrier peptide (residues 47–57) via cysteine S–S bond by Anaspec, San. The primary sequence of the C2/V1 domain of ⑀PKC and the peptides derived from those regions are provided. After stimulasteric activator derived from a sequence implicated in auto- tion, cells were washed with cold phosphate-buffered saline, inhibitory interactions [12]. Both peptides represent regions scraped in homogenization buffer, passed through a syringe in ⑀C2 that were well conserved in evolution and are differ- needle (25-gauge 5/8-inch), and spun at 100,000 ϫ g for 30 ent enough from the sequences in other PKC isozymes. Antibodies against ⑀PKC were obtained from Santa corresponding to short sequences within the C2 domain Cruz Biotechnology (Santa Cruz, CA) and used at 1:500 diluwith isozyme-selective activities [42] suggests that other tion. TAT-conjugated peptides, ⑀PKC C2-derived peptides, and controls used in the different assays
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