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Ca2+ modulation of cis-unsaturated fatty acid-induced mutant protein kinase C activity: indication of inhibitory Ca2+-binding site in protein kinase C-alpha.

ABSTRACT: The C2 domain in protein kinase C (PKC) is homologous to equivalent domains in a number of important cytoplasmic proteins. Except for its implied function in Ca2+ and phospholipid binding, the precise role of this domain is not well understood. We examined the role of the C2 domain of PKC-alpha using a mutant enzyme in which 80% of this domain has been deleted. This mutant can be activated by phospholipid and diacylglycerol, but is independent of Ca2+. In this regard, its characteristics are similar to those of the novel PKCs (nPKCs), consistent with the notion that the C2 domain of PKC confers its Ca2+ responsiveness. However, when the C2 deletion mutant is activated by cis-unsaturated fatty acid, the activity is strongly inhibited by Ca2+ at micromolar concentrations. The Ca2+ inhibition is dose-dependent and is specific to cis-unsaturated fatty acids. The deletion mutant can also be activated synergistically by diacylglycerol and cis-fatty acid, but again activation is inhibited by Ca2+. Our results indicate that a PKC lacking the C2 domain is Ca2+-responsive and there exists an additional site for Ca2+ that modulates the sensitivity of the enzyme to cis-unsaturated fatty acid but not to diacylglycerol. This modulatory Ca2+-binding site appears to be suppressed by the C2 domain because the presence of the domain reverses the direction of PKC activity induced by cis-unsaturated fatty acid. These results suggest that the modulatory Ca2+-binding site could act as a molecular switch selective for fatty acid activation by sensing the changes in the Ca2+ levels in a cell, serving a possible mechanism of differential activation of cPKC with a C2 domain and nPKC lacking this domain.

SUBMITTER: Eshete F

PROVIDER: S-EPMC1219575 | biostudies-other | 1998 Jul

REPOSITORIES: biostudies-other

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Project description:The cellular response to environmental signals is largely dependent upon the induction of responsive protein kinase signaling pathways. Within these pathways, distinct protein-protein interactions play a role in determining the specificity of the response through regulation of kinase function. The interferon-induced serine/threonine protein kinase, PKR, is activated in response to various environmental stimuli. Like many protein kinases, PKR is regulated through direct interactions with activator and inhibitory molecules, including P58IPK, a cellular PKR inhibitor. P58IPK functions to represses PKR-mediated phosphorylation of the eukaryotic initiation factor 2alpha subunit (eIF-2alpha) through a direct interaction, thereby relieving the PKR-imposed block on mRNA translation and cell growth. To further define the molecular mechanism underlying regulation of PKR, we have utilized an interaction cloning strategy to identify a novel cDNA encoding a P58IPK-interacting protein. This protein, designated P52rIPK, possesses limited homology to the charged domain of Hsp90 and is expressed in a wide range of cell lines. P52rIPK and P58IPK interacted in a yeast two-hybrid assay and were recovered as a complex from mammalian cell extracts. When coexpressed with PKR in yeast, P58IPK repressed PKR-mediated eIF-2alpha phosphorylation, inhibiting the normally toxic and growth-suppressive effects associated with PKR function. Conversely, introduction of P52rIPK into these strains resulted in restoration of both PKR activity and eIF-2alpha phosphorylation, concomitant with growth suppression due to inhibition of P58IPK function. Furthermore, P52rIPK inhibited P58IPK function in a reconstituted in vitro PKR-regulatory assay. Our results demonstrate that P58IPK is inhibited through a direct interaction with P52rIPK which, in turn, results in upregulation of PKR activity. Taken together, our data describe a novel protein kinase-regulatory system which encompasses an intersection of interferon-, stress-, and growth-regulatory pathways.

Project description:BackgroundCa2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling.Methodology and principal findingsFive week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity.Conclusions/significanceGender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts. Therefore, CaMKP could be considered as an important novel cellular target for the development of new therapeutic strategies for heart diseases.

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Ca2+ modulation of cis-unsaturated fatty acid-induced mutant protein kinase C activity: indication of inhibitory Ca2+-binding site in protein kinase C-alpha.

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