Tyrosine Phosphorylation Modifies Protein Kinase C δ-dependent Phosphorylation of Cardiac Troponin I

Abstract

PKC␦ leads to depressed maximum tension and cross-bridge kinetics, attributable to a dominant effect of cTnI-Thr144 phosphorylation. Our data implicate PKC␦-Tyr 311 /Thr 505 phosphorylation as dynamically regulated modifications that alter PKC␦ enzymology and allow for stimulus-specific control of cardiac mechanics during growth factor stimulation and oxidative stress. Protein kinase C␦ (PKC␦)2 is a ubiquitous serine/threonine kinase implicated in a wide range of cellular responses (1, 2). PKC␦ is conventionally viewed as a lipid-dependent enzyme that is anchored to membranes in close proximity to target substrates through interactions with lipid cofactors. However, there is recent evidence that PKC␦ also is dynamically regulated through activation loop (Thr 505 ) phosphorylation (3, 4). For other PKC isoforms, activation loop phosphorylation is a stable "priming" phosphorylation completed during de novo enzyme synthesis (5). In the case of cPKCs, activation loop phosphorylations are mediated by phosphoinositide-dependent kinase-1 and are essential to generate a catalytically competent enzyme. Although newly synthesized PKC␦ also undergoes maturational phosphoinositide-dependent kinase-1-dependent Thr 505 phosphorylation, PKC␦ differs from other PKC isoforms in that 1) PKC␦ is a catalytically active enzyme even without Thr 505 phosphorylation and 2) PKC␦-Thr 505 phosphorylation is dynamically regulated through an autocatalytic mechanism (4). Although there are hints that Thr 505 phosphorylation might contribute to the control of PKC␦ enzymology, a PKC␦-Thr 505 autophosphorylation mechanism that regulates the actions of PKC␦ toward a physiologically relevant substrate in a differentiated cell has never been reported.PKC␦ also is regulated through tyrosine phosphorylation. However, the consequences of PKC␦ tyrosine phosphorylation remain disputed, because PKC␦ tyrosine phosphorylation is variably linked to increased, decreased, or unchanged PKC␦ activity (1). Inconsistencies in the literature have been attributed to the presence of multiple tyrosine residues throughout the structure of PKC␦ (including

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…Cantrell and colleagues have also recently shown that deletion of PDK-1 in thymocytes does not abolish PKC A-loop phosphorylation [132], while the enzyme has also been reported to autophosphorylate its own A-loop in cardiomyocytes via a mechanism that is dependent on prior tyrosine phosphorylation of the enzyme at the hinge domain by Src kinases [46,32]. These studies imply that autophosphorylation or indeed a kinase other than PDK-1 may catalyse phosphorylation at this site on PKC .…”

“…Phosphorylation of PKC at Tyr-313 by Src family kinases has also been reported in a variety of cell types in response to non-apoptotic stimuli [117][118][119]46,120,32,121,100]. While some of these studies have shown that phosphorylation at Tyr-313 is required for PKC function, one consequence of Tyr-313 phosphorylation is that it regulates the ability of the enzyme to autophosphorylate its own A-loop and thus fine-tune substrate specificity [32,46].…”

“…Mutation of the A-loop residue of PKC has no effect on in-vitro catalytic activity [30,31,13]. Instead, phosphorylation of this residue on PKC fine-tunes the specificity of the enzyme towards some of its substrates [13,32]. Molecular modelling and mutational analysis have demonstrated that in the absence of A-loop phosphorylation, PKC catalytic activity is stabilised by a pair of phenylalanine residues and an additional 20 residues that are located Nterminal to the kinase domain [13].…”

“…While some of these studies have shown that phosphorylation at Tyr-313 is required for PKC function, one consequence of Tyr-313 phosphorylation is that it regulates the ability of the enzyme to autophosphorylate its own A-loop and thus fine-tune substrate specificity [32,46]. It is also likely that interdependency exists between the A-loop and Tyr-313 sites on PKC , as mutation of the A-loop residue to a non-phosphorylatable residue inhibits Tyr-313 phosphorylation [122].…”

The role of protein kinase C-mediated phosphorylation of sarcomeric proteins in the heart—detrimental or beneficial?

“…Serines 23 and 24, which are located in the cardiac specific N-terminal domain (residues 1-30) of cTnI (absent in skeletal TnI) are the main targets of PKA (Kooij et al 2010b;Solaro 2008). It has recently became evident that these sites can also be cross-phosphorylated by PKCδ, PKCα, PKCβ, PKG, and PKD (Table 1) (Haworth et al 2004;Layland et al 2002Layland et al , 2005Swiderek et al 1990;Sumandea et al 2008). It has been generally accepted that phosphorylation of these sites is an important mechanism for increasing the rate of relaxation during β-adrenergic receptor stimulation in the heart.…”