Protein kinase C mechanisms that contribute to cardiac remodelling

Abstract

Protein phosphorylation is a highly-regulated and reversible process that is precisely controlled by the actions of protein kinases and protein phosphatases. Factors that tip the balance of protein phosphorylation lead to changes in a wide range of cellular responses, including cell proliferation, differentiation and survival. The protein kinase C (PKC) family of serine/threonine kinases sits at nodal points in many signal transduction pathways; PKC enzymes have been the focus of considerable attention since they contribute to both normal physiological responses as well as maladaptive pathological responses that drive a wide range of clinical disorders. This review provides a background on the mechanisms that regulate individual PKC isoenzymes followed by a discussion of recent insights into their role in the pathogenesis of diseases such as cancer. We then provide an overview on the role of individual PKC isoenzymes in the regulation of cardiac contractility and pathophysiological growth responses, with a focus on the PKC-dependent mechanisms that regulate pump function and/or contribute to the pathogenesis of heart failure.

Keywords: myocardial remodelling; post translational modification; protein kinase C.

Figure 1. The domain structure of PKC family enzymes

(A) Schematic showing pseudosubstrate (red rectangle), C1 domain (orange rectangle; atypical C1 domain is hatched to indicate it is not a DAG sensor), C2 domain (yellow rectangle; novel C2 domain is hatched to indicate it is not a Ca²⁺-driven plasma membrane sensor), connecting hinge segment, kinase domain (cyan) and C-terminal tail (CT, grey rectangle). Also indicated are the Y/W switch (purple circle) in the C1B domain that dictates affinity for DAG-containing membranes and the three priming phosphorylations in the kinase domain and CT (note atypical PKC isoenzymes have Glu at phospho-acceptor position of hydrophobic motif). Atypical PKCs have a PBI domain that mediates their interaction with protein scaffolds. Table on right shows dependence of PKC family members on C1 domain cofactors, DAG and phosphatidylserine (PS) and C2 domain cofactors, Ca²⁺ and PIP₂. Adapted from [112]: Antal, C.E. and Newton, A.C. (2014) Tuning the signaling output of protein kinase C. Biochem. Soc. Trans. 42, 1477–1483. (B) Reinterpreted structure of PKCβII [38] from lattice packing [37] showing intramolecular autoinhibition by the C2 domain (yellow), which clamps over the kinase domain (cyan), interfacing also with the C-terminal tail (grey). Also shown is the pseudosubstrate (red), which was modelled into the substrate-binding cavity. Adapted from [38]: Antal, C.E., Callender, J.A., Kornov, A.P., Taylor, S.S. and Newton, A.C. (2015) Intramolecular C2 domain-mediated autoinhibition of protein kinase CβII. Cell Rep. 12, 1252–1260.

Figure 2. Model for regulation of conventional PKC by phosphorylation and second messengers

(A) Unprimed PKCβII is in a membrane-associated, open conformation in which its C1A, C1B and C2 domains are fully exposed and the pseudosubstrate and C-terminal tail are unmasked. (B) Upon priming phosphorylation at its activation loop (Thr⁵⁰⁰, magenta) by PDK-1, followed by autophosphorylation at the turn motif (Thr⁶⁴¹, orange) and the hydrophobic motif (Ser⁶⁶⁰, green), PKCβII adopts a closed conformation in which the C2 domain interfaces with the kinase domain and traps the pseudosubstrate into the substrate-binding site, both C1 domains become masked, and the primed enzyme localizes to the cytosol. This autoinhibition and masking of second messenger sensors ensures efficient suppression of activity in the absence of appropriate stimuli. (C) In response to agonists that promote PIP₂ hydrolysis, Ca²⁺ binds the C2 domain of cytosolic PKCβII via a low affinity interaction such that upon the next diffusion-controlled membrane encounter, the Ca²⁺-bound C2 domain is retained at the plasma membrane via Ca²⁺-bridging to anionic lipids and binding to PIP₂. This rearrangement of the C2 domain is accompanied by unmasking of the hinge connecting the C2 domain and the kinase domain. (D) Membrane-targeted PKC binds the membrane-embedded ligand, DAG, predominantly via the C1B domain, resulting in release of the pseudosubstrate from the substrate-binding cavity, thereby activating PKC. Only one of the C1 domains binds DAG in the membrane at a time. (E) Activated PKC is in a conformation in which the phosphorylation sites are exposed, resulting in dephosphorylation at the hydrophobic motif by PHLPP and at the turn motif and activation loop by okadaic-sensitive phosphatases such as PP2A. The dephosphorylated enzyme is shunted to degradation. However, binding of Hsp70 to the dephosphorylated turn motif permits PKC to become rephosphorylated and re-enter the pool of signalling-competent PKC.

Figure 3. Non-canonical PKC activation mechanisms: proteolytic cleavage of PKCα and tyrosine phosphorylation of PKCδ

Left: Calpain cleavage of PKCα at a site in the hinge region results in the release of a catalytically active kinase domain fragment that targets to the nucleus and induces pathological cardiac remodelling. Right: PKCδ is recovered from resting cardiomyocytes as a Ser³⁵⁷-phosphorylated/lipid-dependent serine kinase; the pink circle denotes phosphorylation at Ser³⁵⁷ in the ATP-positioning G-loop in the kinase domain. Oxidative stress leads to the activation of Src family kinases and the phosphorylation of PKCδ at Tyr³¹¹ (denoted by the purple circle). This generates a docking site for the phosphotyrosine binding C2 domain, results in a C2 domain–pTyr³¹¹ interaction that induces long-range conformational changes that culminate in Ser³⁵⁷ dephosphorylation. PKCδ is converted into a lipid-independent serine/threonine kinase as a result of the decrease in G-loop Ser³⁵⁷ phosphorylation.