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Formation of protein kinase C(epsilon)-Lck signaling modules confers cardioprotection.

ABSTRACT: The epsilon isoform of protein kinase C (PKCepsilon) is a member of the PKC family of serine/threonine kinases and plays a critical role in protection against ischemic injury in multiple organs. Functional proteomic analyses of PKCepsilon signaling show that this isozyme forms multiprotein complexes in the heart; however, the precise signaling mechanisms whereby PKCepsilon orchestrates cardioprotection are poorly understood. Here we report that Lck, a member of the Src family of tyrosine kinases, forms a functional signaling module with PKCepsilon. In cardiac cells, PKCepsilon interacts with, phosphorylates, and activates Lck. In vivo studies showed that cardioprotection elicited either by cardiac-specific transgenic activation of PKCepsilon or by ischemic preconditioning enhances the formation of PKCepsilon-Lck modules. Disruption of these modules, via ablation of the Lck gene, abrogated the infarct-sparing effects of these two forms of cardioprotection, indicating that the formation of PKCepsilon-Lck signaling modules is required for the manifestation of a cardioprotective phenotype. These findings demonstrate, for the first time to our knowledge, that the assembly of a module (PKCepsilon-Lck) is an obligatory step in the signal transduction that results in a specific phenotype. Thus, PKCepsilon-Lck modules may serve as novel therapeutic targets for the prevention of ischemic injury.

SUBMITTER: Ping P

PROVIDER: S-EPMC150872 | biostudies-literature | 2002 Feb

REPOSITORIES: biostudies-literature

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Formation of protein kinase C(epsilon)-Lck signaling modules confers cardioprotection.

Ping Peipei P Song Changxu C Zhang Jun J Guo Yiru Y Cao Xinan X Li Richard C X RC Wu Wenjian W Vondriska Thomas M TM Pass Jason M JM Tang Xian-Liang XL Pierce William M WM Bolli Roberto R

The Journal of clinical investigation 20020201 4

The epsilon isoform of protein kinase C (PKCepsilon) is a member of the PKC family of serine/threonine kinases and plays a critical role in protection against ischemic injury in multiple organs. Functional proteomic analyses of PKCepsilon signaling show that this isozyme forms multiprotein complexes in the heart; however, the precise signaling mechanisms whereby PKCepsilon orchestrates cardioprotection are poorly understood. Here we report that Lck, a member of the Src family of tyrosine kinases ...[more]

PMID: 11854322

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Project description:The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases, Lck. Upon T-cell antigen receptor (TCR) triggering, Lck kinase activity induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process via their capacity to interact with kinases and regulate their intracellular translocations. An adaptor protein, receptor for activated C kinase 1 (RACK1), was chosen as a viable option, and its capacity to bind Lck and aid the process of activation-induced redistribution of Lck was assessed. Our microscopic observation showed that T-cell activation induces a rapid, concomitant, and transient co-redistribution of Lck and RACK1 into the forming immunological synapse. Consistent with this observation, the formation of transient RACK1-Lck complexes were detectable in primary CD4+ T-cells with their maximum levels peaking 10 s after TCR-CD4 co-aggregation. Moreover, RACK1 preferentially binds to a pool of kinase active pY394Lck, which co-purifies with high molecular weight cellular fractions. The formation of RACK1-Lck complexes depends on functional SH2 and SH3 domains of Lck and includes several other signaling and cytoskeletal elements that transiently bind the complex. Notably, the F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence of kinase active Lck suggesting that the formation of RACK1-pY394Lck-α-actinin-1 complex serves as a signal module coupling actin cytoskeleton bundling with productive TCR/CD4 triggering. In addition, the treatment of CD4+ T-cells with nocodazole, which disrupts the microtubular network, also blocked the formation of RACK1-Lck complexes. Importantly, activation-induced Lck redistribution was diminished in primary CD4+ T-cells by an adenoviral-mediated knockdown of RACK1. These results demonstrate that in T cells, RACK1, as an essential component of the multiprotein complex which upon TCR engagement, links the binding of kinase active Lck to elements of the cytoskeletal network and affects the subcellular redistribution of Lck.

Project description:AimsHemodynamic shear stress participates in maintaining vascular redox status. Elucidating flow-mediated endothelial metabolites enables us to discover metabolic biomarkers and therapeutic targets. We posited that flow-responsive vascular endothelial growth factor receptor (VEGFR)-protein kinase C isoform epsilon (PKC?)-6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) signaling modulates glycolytic metabolites for vascular repair.ResultsBidirectional oscillatory flow (oscillatory shear stress [OSS]: 0.1?±?3 dyne·cm-2 at 1?Hz) upregulated VEGFR-dependent PKC? expression to a greater degree than did unidirectional pulsatile flow (pulsatile shear stress [PSS]: 23?±?8 dyne·cm-2 at 1?Hz) in human aortic endothelial cells (p?<?0.05, n?=?3). PSS and OSS further upregulated PKC?-dependent PFKFB3 expression for glycolysis (p?<?0.05, n?=?4). Constitutively active PKC? increased, whereas dominant-negative PKC? reduced both basal and maximal extracellular acidification rates for glycolytic flux (p?<?0.01, n?=?4). Metabolomic analysis demonstrated an increase in PKC?-dependent glycolytic metabolite, dihydroxyacetone (DHA), but a decrease in gluconeogenic metabolite, aspartic acid (p?<?0.05 vs. control, n?=?6). In a New Zealand White rabbit model, both PKC? and PFKFB3 immunostaining was prominent in the PSS- and OSS-exposed aortic arch and descending aorta. In a transgenic Tg(flk-1:EGFP) zebrafish model, GATA-1a morpholino oligonucleotide injection (to reduce viscosity-dependent shear stress) impaired vascular regeneration after tail amputation (p?<?0.01, n?=?20), which was restored with PKC? messenger RNA (mRNA) rescue (p?<?0.05, n?=?5). As a corollary, siPKC? inhibited tube formation and vascular repair, which were restored by DHA treatment in our Matrigel and zebrafish models. Innovation and Conclusion: Flow-sensitive VEGFR-PKC?-PFKFB3 signaling increases the glycolytic metabolite, dihydroxyacetone, to promote vascular repair. Antioxid. Redox Signal. 28, 31-43.

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Formation of protein kinase C(epsilon)-Lck signaling modules confers cardioprotection.

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Formation of protein kinase C(epsilon)-Lck signaling modules confers cardioprotection.

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