Formation of protein kinase Cε-Lck signaling modules confers cardioprotection
Peipei Ping, … , William M. Pierce, Roberto Bolli
Peipei Ping, … , William M. Pierce, Roberto Bolli
Published February 15, 2002
Citation Information: J Clin Invest. 2002;109(4):499-507. https://doi.org/10.1172/JCI13200.
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Article

Formation of protein kinase Cε-Lck signaling modules confers cardioprotection

Abstract

The ε isoform of protein kinase C (PKCε) 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 PKCε signaling show that this isozyme forms multiprotein complexes in the heart; however, the precise signaling mechanisms whereby PKCε 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 PKCε. In cardiac cells, PKCε interacts with, phosphorylates, and activates Lck. In vivo studies showed that cardioprotection elicited either by cardiac-specific transgenic activation of PKCε or by ischemic preconditioning enhances the formation of PKCε-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 PKCε-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 (PKCε-Lck) is an obligatory step in the signal transduction that results in a specific phenotype. Thus, PKCε-Lck modules may serve as novel therapeutic targets for the prevention of ischemic injury.

Authors

Peipei Ping, Changxu Song, Jun Zhang, Yiru Guo, Xinan Cao, Richard C.X. Li, Wenjian Wu, Thomas M. Vondriska, Jason M. Pass, Xian-Liang Tang, William M. Pierce, Roberto Bolli

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Figure 1

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PKCε exhibits direct physical interactions with Lck in vivo and in vitro...
PKCε exhibits direct physical interactions with Lck in vivo and in vitro. (a) Lck co-resided with PKCε (upper panel) but not with PKCη (lower panel). Upper panel: 200 μg proteins were immunoprecipitated with either PKCε mAb’s (lanes 1 and 2) or IgG (lanes 3 and 4) and immunoblotted with Lck antibodies. Lane 5 served as positive control. Lower panel: 200 μg proteins were immunoprecipitated with the PKCη polyclonal antibodies and immunoblotted with Lck antibodies (lanes 1–4). Lane 5 served as positive control. (b) Mouse myocardial Lck interacted with GST-PKCε. Lck expression was detected in cardiac tissue by GST-PKCε pull-down (lane 1). GST-null vector was negative control (lane 2). Lane 3 served as positive control. (c) PKCε exhibited physical interactions with Lck in vitro. Recombinant GST-PKCε proteins were incubated with in vitro translated and [35S]methionine-labeled Lck. Lanes 1–3 contain GST-PKCε and Lck with various concentrations of NaCl; lane 4 served as negative control without Lck; lane 5 served as the [35S]methionine-labeled Lck (positive control). (d) Lck interacted with both the catalytic (CHA) and the regulatory (RHA) domains of PKCε. GST-Lck proteins were incubated with in vitro translated and [35S]methionine-labeled CHA and RHA proteins. Lanes 1–5 depict GST-Lck pull-down of CHA with various concentrations of NaCl; lane 6 served as positive control (CHAproteins); lanes 7–11 depict GST-Lck pull-down of RHA with various concentrations of NaCl; lane 12 served as positive control (RHAproteins). (e) ELISA-based binding assays were performed to determine Lck interactions with CHA and RHA. GST-Lck was found to bind to CHA proteins (left) and RHA proteins (right) with similar affinity.