The atypical protein kinase C (PKC) member PKC-has been implicated in several signal transduction pathways regulating differentiation, proliferation or apoptosis of mammalian cells. We report here the identification of a cytoplasmic and membrane-associated protein that we name zetainteracting protein (ZIP) and that interacts with the regulatory domain of PKC-but not classic PKCs. The structural motifs in ZIP include a recently defined ZZ zinc finger as a potential protein binding module, two PEST sequences and a novel putative protein binding motif with the consensus sequence YXDEDX 5 SDEE͞D. ZIP binds to the pseudosubstrate region in the regulatory domain of PKC-and is phosphorylated by PKCin vitro. ZIP dimerizes via the same region that promotes binding to PKC-suggesting a competitive situation between ZIP:ZIP and ZIP:PKC-complexes. In the absence of PKC-proper subcellular localization of ZIP is impaired and we show that intracellular targeting of ZIP is dependent on a balanced interaction with PKC-. Taking into account the recent isolation of ZIP by others in different contexts we propose that ZIP may function as a scaffold protein linking PKC-to protein tyrosine kinases and cytokine receptors.The intracellular propagation of pleiotropic signals is the major field of activity of the protein kinase C (PKC) family. Besides the classic (␣, ␤I, ␤II, ␥) and the novel (␦, , , ) members the PKC family comprises two atypical members (, ͞) that are distinguished structurally by the presence of only a single PKC zinc finger module in their regulatory domain and biochemically by their inability to bind and to respond to phorbol esters and diacylglycerol (1-3). Most cells and tissues express several PKC enzymes suggesting that the members of this family do not have overlapping functions (4). While the classic and novel members are expected to participate in signal transduction from cell surface receptors that trigger the generation of diacylgycerol by activating phospholipases C the mode of activation and the function of the atypical members is much less clear (5, 6). The search for a function has connected PKC-with two distinct signaling pathways as the most attractive sites of action of this enzyme. First, the finding that phosphatidylinositol-3,4,5-trisphosphate can activate PKC-in vitro pointed to the possibility that PKC-participates in phosphorylation events downstream of phosphatidylinositol 3-kinase activation by receptor tyrosine kinases (7). Secondly, PKC-has been proposed as a mediator of the growth inhibitory and apoptotic actions of ceramide, an intracellular messenger generated by hydrolysis of sphingolipids (8). Evidence for effects of the sphingomyelin cycle on PKC-activity, specifically in tumor necrosis factor ␣ signaling, has been presented recently (9-11).In addition, overexpression of PKC-has been shown to be necessary and sufficient to deregulate growth control in mouse fibroblasts supporting a crucial role for PKC-in ras-induced mitogenic signaling (12), albeit conflicting data have been re...

The extracellular matrix glycoprotein tenascin-R (TN-R), colocalizing with hyaluronan, phosphacan, and aggregating chondroitin sulphate proteoglycans in the white and grey matter, is accumulated in perineuronal nets that surround different types of neurons in many brain regions. To characterize the role of TN-R in the formation of perineuronal nets, we studied their postnatal development in wild-type mice and in a TN-R knock-out mutant by using the lectin Wisteria floribunda agglutinin and an antibody to nonspecified chondroitin sulphate proteoglycans as established cytochemical markers. We detected the matrix components TN-R, hyaluronan, phosphacan, neurocan, and brevican in the perineuronal nets of cortical and subcortical regions. In wild-type mice, lectin-stained, immature perineuronal nets were first seen on postnatal day 4 in the brainstem and on day 14 in the cerebral cortex. The staining intensity of these nets for TN-R, hyaluronan, phosphacan, neurocan, and brevican was extremely weak or not distinguishable from that of the surrounding neuropil. However, all markers showed an increase in staining intensity of perineuronal nets reaching maximal levels between postnatal days 21 and 40. In TN-R-deficient animals, the perineuronal nets tended to show a granular component within their lattice-like structure at early stages of development. Additionally, the staining intensity in perineuronal nets was reduced for brevican, extremely low for hyaluronan and neurocan, and virtually no immunoreactivity was detectable for phosphacan. The granular configuration of perineuronal nets became more predominant with advancing age of the mutant animals, indicating the continued abnormal aggregation of chondroitin sulphate proteoglycans complexed with hyaluronan. As shown by electron microscopy in the cerebral cortex, the disruption of perineuronal nets was not accompanied by apparent changes in the synaptic structure on net-bearing neurons. The regional distribution patterns and the temporal course of development of perineuronal nets were not obviously changed in the mutant. We conclude that the lack of TN-R initially and continuously disturbs the molecular scaffolding of extracellular matrix components in perineuronal nets. This may interfere with the development of the specific micromilieu of the ensheathed neurons and adjacent glial cells and may also permanently change their functional properties.

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.

No abstract

No abstract