Cytosolic targeting domains of gamma and delta calmodulin-dependent protein kinase II.

Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II) isozyme variability is the result of alternative usage of variable domain sequences. Isozyme expression is cell type-specific to transduce the appropriate Ca(2+) signals. We have determined the subcellular targeting domain of delta(E) CaMK-II, an isozyme that induces neurite outgrowth, and of a structurally similar isozyme, gamma(C) CaMK-II, which does not induce neurite outgrowth. delta(E) CaMK-II co-localizes with filamentous actin in the perinuclear region and in cellular extensions. In contrast, gamma(C) CaMK-II is uniformly cytosolic. Constitutively active delta(E) CaMK-II induces F-actin-rich extensions, thereby supporting a functional role for its localization. C-terminal constructs, which lack central variable domain sequences, can oligomerize and localize like full-length delta(E) and gamma(C) CaMK-II. Central variable domains themselves are monomeric and have no targeting capability. The C-terminal 95 residues of delta CaMK-II also has no targeting capability but can efficiently oligomerize. These findings define a targeting domain for gamma and delta CaMK-IIs that is in between the central variable and association domains. This domain is responsible for the subcellular targeting differences between gamma and delta CaMK-IIs.

NEU overexpression in the furan rat model of cholangiocarcinogenesis compared with biliary ductal cell hyperplasia.

Immunohistochemical studies have suggested that the tyrosine kinase growth factor receptor p185neu is overexpressed in a high percentage of human cholangiocarcinomas. To establish the specificity and temporal relationship between the expression of this receptor in cholangiocarcinogenesis, we investigated c-neu expression in precancerous cholangiofibrotic tissue and subsequently derived primary and transplantable cholangiocarcinomas originated in the livers of furan-treated rats. Proliferated bile ductules formed in rat models of bile ductular hyperplasia and the cell types of normal adult rat liver were also analyzed for c-neu expression. c-neu expression was not detected in normal adult rat liver by either Western blotting, immunohistochemistry, or in situ hybridization. In comparison, all of the cholangiocarcinomas analyzed, which were characterized by intestinal-type mucin-producing neoplastic glands, exhibited a prominent band with a molecular weight 185 kd, corresponding to p185neu. Only the neoplastic glandular epithelia of the cholangiocarcinomas showed a strong immunoreactivity for p185neu, which was predominantly localized to their cell surface but also observed cytoplasmically. In situ hybridization further revealed the cytoplasm of the tumor glandular epithelial cells to be strongly positive for c-neu mRNA transcripts. Of particular interest was our finding that c-neu is expressed early in furan cholangiocarcinogenesis, being more pronounced in the metaplastic intestinal glands of cholangiofibrotic tissue than in hyperplastic biliary epithelial cells in either the same tissue or in hyperplastic bile ductule tissue. Our results demonstrate that c-neu overexpression is a prominent feature of intestinal-type cholangiocarcinomas as well as of metaplastic intestinal glands that precede their development and is detected at lower levels in hyperplastic biliary epithelia. The overexpression of c-neu in the metaplastic and malignant neoplastic glands also correlated with their increased proliferating cell nuclear antigen (PCNA) labeling indices relative to those of hyperplastic biliary ducts and ductules and also appeared to correlate with their intestinal glandular pattern of differentiation.