Identification of a Novel Phosphorylation Site in Protein Phosphatase Inhibitor-1 as a Negative Regulator of Cardiac Function

Abstract

Human and experimental heart failure is characterized by increases in type-1 protein phosphatase activity, which may be partially attributed to inactivation of its endogenous regulator, protein phosphatase inhibitor-1. Inhibitor-1 represents a nodal integrator of two major second messenger pathways, adenosine 3,5-cyclic monophosphate (cAMP) and calcium, which mediate its phosphorylation at threonine 35 and serine 67, respectively. Here, using recombinant inhibitor-1 wild-type and mutated proteins, we identified a novel phosphorylation site in inhibitor-1, threonine 75. This phosphoamino acid was phosphorylated in vitro by protein kinase C␣ independently and to the same extent as serine 67, the previous protein kinase C␣-identified site. Generation of specific antibodies for the phosphorylated and dephosphorylated threonine 75 revealed that this site is phosphorylated in rat and dog hearts. Adenoviral-mediated expression of the constitutively phosphorylated threonine 75 inhibitor-1 in isolated myocytes was associated with specific stimulation of type-1 protein phosphatase activity and marked inhibition of the sarcoplasmic calcium pump affinity for calcium, resulting in depressed contractility. Thus, phosphorylation of inhibitor-1 at threonine 75 represents a new mechanism of cardiac contractility regulation, partially through the alteration of sarcoplasmic reticulum calcium transport activity.A fine-tuned regulation of protein kinase and protein phosphatase activities is essential in the control of the phosphorylation state of various key phosphoprotein substrates, which modulate glycogen metabolism, protein synthesis, cell division, neuronal signaling, and muscle contraction (1-3). A cross-talk between the second messenger cAMP-dependent protein kinase (PKA) 4 and the type-1 protein phosphatase (PP1) occurs at the level of an endogenous phosphoprotein, inhibitor-1 (I-1), allowing amplification of the cAMP signaling cascade. Inhibitor-1 was first identified in rabbit skeletal muscle (4), but it is widely expressed in mammalian tissues and highly conserved across species (5). This thermostable protein, upon phosphorylation by PKA at Thr-35, potently inhibits PP1, enhancing cAMP-mediated protein phosphorylation (6 -9). In cardiac muscle, the regulation of PP1 by I-1 has been shown to play a major role in both basal contractility (2, 10), as well as in the heart response to ␤-adrenergic stimulation (11, 12). The positive inotropic effect of the ␤-agonist, isoproterenol, is accompanied by I-1 phosphorylation, resulting in inhibition of PP1 activity (13). This decrease in PP1 augments the cAMP-stimulating effects in enhancing cardiac contractility by preventing the dephosphorylation of important proteins involved in regulating the contractile state of the heart. Intriguingly, a constitutively activated form of I-1 (T35D; AA1-65) enhanced cardiac function and the heart response to ␤-adrenergic agonists, and protected the heart from developing hypertrophy induced by pressure overload (10). Furthermore, acute adenovira...

Human Mutation in the Anti-apoptotic Heat Shock Protein 20 Abrogates Its Cardioprotective Effects

“…The P20L mutant was generated using the Quik-Change site-directed mutagenesis II kit (Stratagene). Subsequently, WT and mutant Hsp20 were expressed as glutathione-S-transferase-fusion proteins in BL21 CodonPlus (DE3)-RIPL competent cells as described previously (20). Briefly, the proteins were purified using the B-PER glutathione-S-transferase fusion protein purification kit (Pierce), and the glutathione-S-transferase tag was digested using the PreScission protease (GE Healthcare).…”

“…The protein concentration was determined using the MicroBCA assay (Pierce). The adenoviruses were generated as previously described (20). Briefly, the Ad-Easy XL system (Stratagene) was used to generate adenoviruses expressing the WT-Hsp20 (Ad.WT-Hsp20), the Hsp20 mutant (Ad.P20L-Hsp20), or green fluorescent protein (Ad.GFP).…”

“…Myocytes from adult male Sprague-Dawley rats (ϳ300 g) were isolated by collagenase digestion as previously described (20). Myocytes were resuspended in modified culture medium (M199, Sigma), counted, and plated on laminin-coated plates or dishes for 2 h at 37°C in a humidified, 5% CO 2 incubator and subsequently infected with the adenoviruses at a multiplicity of infection of 500 for 2 h. At 48 h post-infection, the cells were subjected to simulated ischemia/reperfusion.…”

“…At basal levels, no phosphorylated Hsp20 could be detected, consistent with the low activity of PKA in isolated myocytes. To test the specificity of this antibody, peptide competition was performed with the anti- [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] in human, dog, rat, and mouse shows that proline 20 is fully conserved. B, secondary structure predictions revealed alterations in the helical content around the Ser 16 phosphorylation site as well as around the site of the mutation.…”

Cytoskeletal role in protection of the failing heart by β-adrenergic blockade

“…It should be clear that the data in this study do not constitute a general mechanism explaining the entirety of the well-established beneficial effects of chronic ␤-adrenergic blockade in pathological myocardial states (38), which are likely based to some extent on preventing the more general cytotoxic cardiomyocyte effects such as we initially observed with sustained ␤-adrenergic stimulation (32). As an example, ␤-blockade-mediated reductions in phosphatase activity could increase Ca 2ϩ cycling protein phosphorylation, thus enhancing inotropism via effects on the rate and amplitude of the Ca 2ϩ transient (41). Instead, the data reported here are but one specific, concrete example of the more general improvement in biological function of the cardiac myocyte by ␤-blockade predicted some time ago (18).…”