Tissue and cellular distribution of the extended family of protein kinase C isoenzymes.

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.

Selective regulation of expression of protein kinase C beta isoenzymes occurs via alternative splicing.

The mechanisms involved in regulating the selective expression of protein kinase C (PKC) isoenzymes are poorly understood. Two human B lymphoblastoid cell lines, IM-9 and BJA-B, exhibited differential expression of the two alternatively spliced products of the PKC beta gene, PKC beta I and beta II. The IM-9 cell line expressed 3-4-fold more PKC beta II protein than the BJA-B cell line, whereas the BJA-B cell line expressed 2-3-fold more PKC beta I protein. This differential expression was found to be regulated at the mRNA level. Comparison of PKC beta I and beta II messages in poly(A)+ mRNA and total cellular RNA revealed that selective polyadenylation was not involved. The messages for PKC beta I and beta II had comparable half-lives in both cell lines, ruling out differential message stability. In addition, similar ratios of PKC beta I and beta II messages in cytosolic and nuclear fractions suggested that differential mRNA transport was not involved. In the IM-9 cell line, the predominance of mature PKC beta II message as well as that of a larger message spliced to PKC beta II provided evidence that the differential expression of PKC beta II was regulated at the level of mRNA splicing. In the BJA-B cell line, equal amounts of mature PKC beta I and beta II message and the absence of the larger message suggested that the splicing of the PKC beta gene product can be regulated to produce altered ratios of PKC beta I and beta II. Implications of these studies on the differential expression of PKC isoenzymes and their roles in biology are discussed.