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. 2001 Sep 11;98(20):11114–11119. doi: 10.1073/pnas.191369098

Figure 1.

Figure 1

Rational design of δPKC translocation inhibitor and activator. (A) Alignment of the primary sequence of rat δPKC and mouse θPKC V1 domains (Protein Data Bank accession nos. KIRTCD and NP_032885, respectively); shadowed boxes indicate identity. Location of β-strands and the α-helix based on δV1 structure analysis (26) are indicated below the sequence; sequences most different between the two isozymes are marked above with a color bracket. (B) The secondary structure of δV1 (26) (Lower) and a modeled secondary structure of ɛV1 (G.C., D.M.-R., and L.B., unpublished work; Upper) are schematically depicted, according to ref. 32. Numbering of β-strands in δV1 and ɛV1 domains are marked as in A for δV1. The sequence corresponding to δV1–1, amino acids 8–17 [SFNSYELGSL], δV1–2, amino acids 35–45 [ALTTDRGKTLV], and ψδRACK, amino acids 74–81 [MRAAEDPM], are marked as in A, in red, yellow, and green, respectively. The sequence corresponding to the ɛPKC-selective inhibitor peptide, ɛV1–2, amino acids 14–21 [EAVSLKPT] (12), and activator peptide, ψɛRACK, amino acids 85–92 [HDAPIGYD] (13), are marked in red and green, respectively (Upper). (C) Crystal structure of the δV1 domain (Protein Data Bank ID no. 1BDY; ref. 26) is depicted with areas marked in colors corresponding to those in A and B. (D) Western blot analysis of cytosolic and particulate fractions from adult rat cardiac myocytes was carried out as described (13) to demonstrate isozyme-selective effects on δPKC translocation. Cells were treated with PMA in the presence and absence of δV1–1. (Left) Autoradiogram of soluble (S) and particulate (P) fractions probed with anti-δPKC (Upper) and the same blot probed with anti-ɛPKC antibodies (Lower). (Right) Mean ± SEM of data from three experiments; translocation is expressed as the amount of each isozyme in the particulate fraction over the amount of that isozyme in nontreated cells. *, P < 0.05; NS, not significant; n = 3. (E) Same as in D, except cells were treated with PMA or ψδRACK and translocation of δPKC and αPKC is shown. **, P < 0.01. (F) Same as D, except cells were treated with δV1–1 in the presence and absence of ψδRACK. **, P < 0.01.