AMP-activated protein kinase phosphorylates cardiac troponin I at Ser-150 to increase myofilament calcium sensitivity and blunt PKA-dependent function

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme central to the regulation of metabolic homeostasis. In the heart AMPK is activated during cardiac stress-induced ATP depletion and functions to stimulate metabolic pathways that restore the AMP/ATP balance. Recently it was demonstrated that AMPK phosphorylates cardiac troponin I (cTnI) at Ser-150 in vitro. We sought to determine if the metabolic regulatory kinase AMPK phosphorylates cTnI at Ser-150 in vivo to alter cardiac contractile function directly at the level of the myofilament. Rabbit cardiac myofibrils separated by two-dimensional isoelectric focusing subjected to a Western blot with a cTnI phosphorylation-specific antibody demonstrates that cTnI is endogenously phosphorylated at Ser-150 in the heart. Treatment of myofibrils with the AMPK holoenzyme increased cTnI Ser-150 phosphorylation within the constraints of the muscle lattice. Compared with controls, cardiac fiber bundles exchanged with troponin containing cTnI pseudo-phosphorylated at Ser-150 demonstrate increased sensitivity of calcium-dependent force development, blunting of both PKA-dependent calcium desensitization, and PKA-dependent increases in length dependent activation. Thus, in addition to the defined role of AMPK as a cardiac metabolic energy gauge, these data demonstrate AMPK Ser-150 phosphorylation of cTnI directly links the regulation of cardiac metabolic demand to myofilament contractile energetics. Furthermore, the blunting effect of cTnI Ser-150 phosphorylation cross-talk can uncouple the effects of myofilament PKA-dependent phosphorylation from β-adrenergic signaling as a novel thin filament contractile regulatory signaling mechanism.     

Cardiac myosin light chain kinase is necessary for myosin regulatory light chain phosphorylation and cardiac performance in vivo

In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca(2+)/calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo.     

Unambiguous characterization of site-specific phosphorylation of leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) in Toll-like receptor 4 (TLR4)-mediated signaling

In the TLR4 signaling pathways, we previously characterized a signal regulator, LRRFIP2, that modulates the time course-dependent changes in NF-κB activity through its dynamic interaction with the TLR adaptor protein, MyD88. However, little is known about the driving force behind the LPS-inducible dynamics between LRRFIP2 and MyD88. We have therefore designed a multiplex label-free quantitative proteomics method to investigate dynamic changes of LRRFIP2 phosphorylation upon LPS stimulation. Given our observation that LRRFIP2 binds to MyD88 through its serine-rich domain in which most of serine residues have the propensity to be phosphorylated, we used collision-activated dissociation- and electron transfer dissociation-based methods in a complementary manner to unambiguously localize phosphorylation sites in the peptides constituting the serine-rich domain. Among 23 phosphorylation sites identified and first quantified by the label-free approach and then verified by the AACT/SILAC (amino acid-coded tagging/stable isotope labeling in cell culture)-based quantitation method, phosphorylation at serine 202 showed a significant LPS-induced dynamic change during the full-course cellular response to LPS stimulation. The substitution of serine 202 with nonphosphorylated residues by site-directed mutagenesis resulted in a weakened LRRFIP2-MyD88 interaction and a concurrently reduced activity in downstream NF-κB. Taking these results together, phosphorylation at serine 202 was found to regulate the dynamics of the LRRFIP2-MyD88 interaction, which in turn modulated the strength and duration of TLR4 signaling. Strategically, we have demonstrated the importance of precise identification of the biologically relevant phosphorylation site(s) using comprehensive mass spectrometry-based quantitative proteomics approaches in guiding downstream biological characterization experiments, which could otherwise be both time- and cost-consuming for a large number of phosphorylation possibilities.     

Cardiac troponin T, a sarcomeric AKAP, tethers protein kinase A at the myofilaments

Efficient and specific phosphorylation of PKA substrates, elicited in response to β-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A-kinase anchoring protein), cardiac troponin T (cTnT). Using yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cTn exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show that PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.     

Phosphorylation of Dopamine Transporter Serine 7 Modulates Cocaine Analog Binding

As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (−)-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states.     

Endosomal targeting of the phosphoinositide 3-phosphatase MTMR2 is regulated by an N-terminal phosphorylation site

MTMR2 is a member of the myotubularin family of inositol lipid phosphatases, a large protein-tyrosine phosphatase subgroup that is conserved from yeast to humans. Furthermore, the peripheral neuromuscular disease Charcot-Marie Tooth disease type 4B has been attributed to mutations in the mtmr2 gene. Because the molecular mechanisms regulating MTMR2 have been poorly defined, we investigated whether reversible phosphorylation might regulate MTMR2 function. We used mass spectrometry-based methods to identify a high stoichiometry phosphorylation site on serine 58 of MTMR2. Phosphorylation at Ser(58), or a phosphomimetic S58E mutation, markedly decreased MTMR2 localization to endocytic vesicular structures. In contrast, a phosphorylation-deficient MTMR2 mutant (S58A) displayed constitutive localization to early endocytic structures. This localization pattern was accompanied by displacement of a PI(3)P-specific sensor protein and an increase in signal transduction pathways. Thus, MTMR2 phosphorylation is likely to be a critical mechanism by which MTMR2 access to its lipid substrate(s) is temporally and spatially regulated, thereby contributing to the control of downstream endosome maturation events.     

Protein Phosphatase 2A Is Regulated by Protein Kinase Cα (PKCα)-dependent Phosphorylation of Its Targeting Subunit B56α at Ser41

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser⁴¹ of B56α. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKCα. We detected a 7-fold higher phosphorylation of B56α in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKCα-phosphorylated B56α. The potency of B56α for PP2A inhibition was markedly increased by PKCα phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56α mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKCα-dependent phosphorylation of B56α was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca²⁺ release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56α. Taken together, our results suggest that PKCα can modify PP2A activity by phosphorylation of B56α at Ser⁴¹. This interplay between PKCα and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca²⁺ homeostasis.     

Structural dynamics of C-domain of cardiac troponin I protein in reconstituted thin filament

The regulatory function of cardiac troponin I (cTnI) involves three important contiguous regions within its C-domain: the inhibitory region (IR), the regulatory region (RR), and the mobile domain (MD). Within these regions, the dynamics of regional structure and kinetics of transitions in dynamic state are believed to facilitate regulatory signaling. This study was designed to use fluorescence anisotropy techniques to acquire steady-state and kinetic information on the dynamic state of the C-domain of cTnI in the reconstituted thin filament. A series of single cysteine cTnI mutants was generated, labeled with the fluorophore tetramethylrhodamine, and subjected to various anisotropy experiments at the thin filament level. The structure of the IR was found to be less dynamic than that of the RR and the MD, and Ca(2+) binding induced minimal changes in IR dynamics: the flexibility of the RR decreased, whereas the MD became more flexible. Anisotropy stopped-flow experiments showed that the kinetics describing the transition of the MD and RR from the Ca(2+)-bound to the Ca(2+)-free dynamic states were significantly faster (53.2-116.8 s(-1)) than that of the IR (14.1 s(-1)). Our results support the fly casting mechanism, implying that an unstructured MD with rapid dynamics and kinetics plays a critical role to initiate relaxation upon Ca(2+) dissociation by rapidly interacting with actin to promote the dissociation of the RR from the N-domain of cTnC. In contrast, the IR responds to Ca(2+) signals with slow structural dynamics and transition kinetics. The collective findings suggested a fourth state of activation.     

The giant protein titin: a regulatory node that integrates myocyte signaling pathways

Titin, the largest protein in the human body, is well known as a molecular spring in muscle cells and scaffold protein aiding myofibrillar assembly. However, recent evidence has established another important role for titin: that of a regulatory node integrating, and perhaps coordinating, diverse signaling pathways, particularly in cardiomyocytes. We review key findings within this emerging field, including those related to phosphorylation of the titin springs, and also discuss how titin participates in hypertrophic gene regulation and protein quality control.     

Delayed phosphorylation of classical protein kinase C (PKC) substrates requires PKC internalization and formation of the pericentrion in a phospholipase D (PLD)-dependent manner

It was previously demonstrated that sustained activation (30-60 min) of protein kinase C (PKC) results in translocation of PKC α and βII to the pericentrion, a dynamic subset of the recycling compartment whose formation is dependent on PKC and phospholipase D (PLD). Here we investigated whether the formation of the pericentrion modulates the ability of PKC to phosphorylate substrates, especially if it reduces substrate phosphorylation by sequestering PKC. Surprisingly, using an antibody that detects phosphosubstrates of classical PKCs, the results showed that the majority of PKC phosphosubstrates are phosphorylated with delayed kinetics, correlating with the time frame of PKC translocation to the pericentrion. Substrate phosphorylation was blocked by PLD inhibitors and was not observed in response to activation of a PKC βII mutant (F663D) that is defective in interaction with PLD and in internalization. Phosphorylation was also inhibited by blocking clathrin-dependent endocytosis, demonstrating a requirement for endocytosis for the PKC-dependent major phosphorylation effects. Serotonin receptor activation by serotonin showed a similar response to phorbol 12-myristate 13-acetate, implicating a potential role of delayed kinetics in G protein-coupled receptor signaling. Evaluation of candidate substrates revealed that the phosphorylation of the PKC substrate p70S6K kinase behaved in a similar manner. Gradient-based fractionation revealed that the majority of these PKC substrates reside within the pericentrion-enriched fractions and not in the plasma membrane. Finally, proteomic analysis of the pericentrion-enriched fractions revealed several proteins as known PKC substrates and/or proteins involved in endocytic trafficking. These results reveal an important role for PKC internalization and for the pericentrion as key determinants/amplifiers of PKC action.     

Regulation of Human Cytidine Triphosphate Synthetase 2 by Phosphorylation

Cytidine triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the K_m and IC₅₀ values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. Metabolic labeling of hCTPS2 with [³²P]H₃PO₄ demonstrated that Ser⁵⁶⁸ and Ser⁵⁷¹ were two major phosphorylation sites, and additional studies demonstrated that Ser⁵⁶⁸ was phosphorylated by casein kinase 1 both in vitro and in vivo. Interestingly, mutation of Ser⁵⁶⁸ (S568A) but not Ser⁵⁷¹ significantly increased hCTPS2 activity, demonstrating that Ser⁵⁶⁸ is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the V_max of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser⁵⁶⁸ as a major site of CTPS2 regulation by phosphorylation.     

MAPK scaffold IQGAP1 binds the EGF receptor and modulates its activation

Cellular responses produced by EGF are mediated through the receptor (EGFR) and by various enzymes and scaffolds. Recent studies document IQGAP1 as a scaffold for the MAPK cascade, binding directly to B-Raf, MEK, and ERK and regulating their activation in response to EGF. We previously showed that EGF is unable to activate B-Raf in cells lacking IQGAP1. However, the mechanism by which IQGAP1 links B-Raf to EGFR was unknown. Here we report that endogenous EGFR and IQGAP1 co-localize and co-immunoprecipitate in cells. EGF has no effect on the association, but Ca(2+) attenuates binding. In vitro analysis demonstrated a direct association mediated through the IQ and kinase domains of IQGAP1 and EGFR, respectively. Calmodulin disrupts this interaction. Using a mass spectrometry-based assay, we show that EGF induces phosphorylation of IQGAP1 Ser(1443), a residue known to be phosphorylated by PKC. This phosphorylation is eliminated by pharmacological inhibition of either EGFR or PKC and transfection with small interfering RNA directed against the PKCα isoform. In IQGAP1-null cells, EGF-stimulated tyrosine phosphorylation of EGFR is severely attenuated. Normal levels of autophosphorylation are restored by reconstituting wild type IQGAP1 and enhanced by an IQGAP1 S1443D mutant. Collectively, these data demonstrate a functional interaction between IQGAP1 and EGFR and suggest that IQGAP1 modulates EGFR activation.     

Novel role for p90 ribosomal S6 kinase in the regulation of cardiac myofilament phosphorylation

In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.     

Activity-dependent phosphorylation of neuronal Kv2.1 potassium channels by CDK5

Dynamic modulation of ion channel expression, localization, and/or function drives plasticity in intrinsic neuronal excitability. Voltage-gated Kv2.1 potassium channels are constitutively maintained in a highly phosphorylated state in neurons. Increased neuronal activity triggers rapid calcineurin-dependent dephosphorylation, loss of channel clustering, and hyperpolarizing shifts in voltage-dependent activation that homeostatically suppress neuronal excitability. These changes are reversible, such that rephosphorylation occurs after removal of excitatory stimuli. Here, we show that cyclin-dependent kinase 5 (CDK5), a Pro-directed Ser/Thr protein kinase, directly phosphorylates Kv2.1, and determines the constitutive level of Kv2.1 phosphorylation, the rapid increase in Kv2.1 phosphorylation upon acute blockade of neuronal activity, and the recovery of Kv2.1 phosphorylation after stimulus-induced dephosphorylation. We also demonstrate that although the phosphorylation state of Kv2.1 is also shaped by the activity of the PP1 protein phosphatase, the regulation of Kv2.1 phosphorylation by CDK5 is not mediated through the previously described regulation of PP1 activity by CDK5. Together, these studies support a novel role for CDK5 in regulating Kv2.1 channels through direct phosphorylation.     

Oncogenic activity of Ect2 is regulated through protein kinase C iota-mediated phosphorylation

The Rho GTPase guanine nucleotide exchange factor Ect2 is genetically and biochemically linked to the PKCι oncogene in non-small cell lung cancer (NSCLC). Ect2 is overexpressed and mislocalized to the cytoplasm of NSCLC cells where it binds the oncogenic PKCι-Par6 complex, leading to activation of the Rac1 small GTPase. Here, we identify a previously uncharacterized phosphorylation site on Ect2, threonine 328, that serves to regulate the oncogenic activity of Ect2 in NSCLC cells. PKCι directly phosphorylates Ect2 at Thr-328 in vitro, and RNAi-mediated knockdown of either PKCι or Par6 leads to a decrease in phospho-Thr-328 Ect2, indicating that PKCι regulates Thr-328 Ect2 phosphorylation in NSCLC cells. Both wild-type Ect2 and a phosphomimetic T328D Ect2 mutant bind the PKCι-Par6 complex, activate Rac1, and restore transformed growth and invasion when expressed in NSCLC cells made deficient in endogenous Ect2 by RNAi-mediated knockdown. In contrast, a phosphorylation-deficient T328A Ect2 mutant fails to bind the PKCι-Par6 complex, activate Rac1, or restore transformation. Our data support a model in which PKCι-mediated phosphorylation regulates Ect2 binding to the oncogenic PKCι-Par6 complex thereby activating Rac1 activity and driving transformed growth and invasion.     

Protein kinase C phosphorylation regulates membrane insertion of GABAA receptor subtypes that mediate tonic inhibition

Tonic inhibition in the brain is mediated largely by specialized populations of extrasynaptic receptors, γ-aminobutyric acid receptors (GABA(A)Rs). In the dentate gyrus region of the hippocampus, tonic inhibition is mediated primarily by GABA(A)R subtypes assembled from α4β2/3 with or without the δ subunit. Although the gating of these receptors is subject to dynamic modulation by agents such as anesthetics, barbiturates, and neurosteroids, the cellular mechanisms neurons use to regulate their accumulation on the neuronal plasma membrane remain to be determined. Using immunoprecipitation coupled with metabolic labeling, we demonstrate that the α4 subunit is phosphorylated at Ser(443) by protein kinase C (PKC) in expression systems and hippocampal slices. In addition, the β3 subunit is phosphorylated on serine residues 408/409 by PKC activity, whereas the δ subunit did not appear to be a PKC substrate. We further demonstrate that the PKC-dependent increase of the cell surface expression of α4 subunit-containing GABA(A)Rs is dependent on Ser(443). Mechanistically, phosphorylation of Ser(443) acts to increase the stability of the α4 subunit within the endoplasmic reticulum, thereby increasing the rate of receptor insertion into the plasma membrane. Finally, we show that phosphorylation of Ser(443) increases the activity of α4 subunit-containing GABA(A)Rs by preventing current run-down. These results suggest that PKC-dependent phosphorylation of the α4 subunit plays a significant role in enhancing the cell surface stability and activity of GABA(A)R subtypes that mediate tonic inhibition.     

Osmotic stress inhibits proteasome by p38 MAPK-dependent phosphorylation

Osmotic stress causes profound perturbations of cell functions. Although the adaptive responses required for cell survival upon osmotic stress are being unraveled, little is known about the effects of osmotic stress on ubiquitin-dependent proteolysis. We now report that hyperosmotic stress inhibits proteasome activity by activating p38 MAPK. Osmotic stress increased the level of polyubiquitinated proteins in the cell. The selective p38 inhibitor SB202190 decreased osmotic stress-associated accumulation of polyubiquitinated proteins, indicating that p38 MAPK plays an inhibitory role in the ubiquitin proteasome system. Activated p38 MAPK stabilized various substrates of the proteasome and increased polyubiquitinated proteins. Proteasome preparations purified from cells expressing activated p38 MAPK had substantially lower peptidase activities than control proteasome samples. Proteasome phosphorylation sites dependent on p38 were identified by measuring changes in the extent of proteasome phosphorylation in response to p38 MAPK activation. The residue Thr-273 of Rpn2 is the major phosphorylation site affected by p38 MAPK. The mutation T273A in Rpn2 blocked the proteasome inhibition that is mediated by p38 MAPK. These results suggest that p38 MAPK negatively regulates the proteasome activity by phosphorylating Thr-273 of Rpn2.     

Removal of the Cardiac Troponin I N-terminal Extension Improves Cardiac Function in Aged Mice

The cardiac troponin I (cTnI) isoform contains a unique N-terminal extension that functions to modulate activation of cardiac myofilaments. During cardiac remodeling restricted proteolysis of cTnI removes this cardiac specific N-terminal modulatory extension to alter myofilament regulation. We have demonstrated expression of the N-terminal-deleted cTnI (cTnI-ND) in the heart decreased the development of the cardiomyopathy like phenotype in a β-adrenergic-deficient transgenic mouse model. To investigate the potential beneficial effects of cTnI-ND on the development of naturally occurring cardiac dysfunction, we measured the hemodynamic and biochemical effects of cTnI-ND transgenic expression in the aged heart. Echocardiographic measurements demonstrate cTnI-ND transgenic mice exhibit increased systolic and diastolic functions at 16 months of age compared with age-matched controls. This improvement likely results from decreased Ca²⁺ sensitivity and increased cross-bridge kinetics as observed in skinned papillary bundles from young transgenic mice prior to the effects of aging. Hearts of cTnI-ND transgenic mice further exhibited decreased β myosin heavy chain expression compared to age matched non-transgenic mice as well as altered cTnI phosphorylation. Finally, we demonstrated cTnI-ND expressed in the heart is not phosphorylated indicating the cTnI N-terminal is necessary for the higher level phosphorylation of cTnI. Taken together, our data suggest the regulated proteolysis of cTnI during cardiac stress to remove the unique cardiac N-terminal extension functions to improve cardiac contractility at the myofilament level and improve overall cardiac function.