Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser²⁰⁵⁶, Thr²⁶⁴⁷ and Thr²⁶⁰⁹ in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser³²⁰⁵ and Thr³⁹⁵⁰ in mitosis. Phosphorylation of Thr³⁹⁵⁰ is DNA-PK-dependent, whereas phosphorylation of Ser³²⁰⁵ requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser³²⁰⁵ in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser³²⁰⁵ in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr⁶⁸ in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs’ role in mitosis may be mechanistically distinct from its well-established role in NHEJ.     

Cross-talk between Rho-associated Kinase and Cyclic Nucleotide-dependent Kinase Signaling Pathways in the Regulation of Smooth Muscle Myosin Light Chain Phosphatase

Ca²⁺ sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr⁶⁹⁷ and/or Thr⁸⁵⁵ (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser⁶⁹⁶ prevents phosphorylation at Thr⁶⁹⁷. However, the effects of Ser⁸⁵⁴ and dual Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser⁶⁹⁶, Thr⁶⁹⁷, Ser⁸⁵⁴, and Thr⁸⁵⁵), Ser phosphorylation events (Ser⁶⁹⁶/Ser⁸⁵⁴) and dual Ser/Thr phosphorylation events (Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵). Dual phosphorylation at Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr⁶⁹⁷ and Thr⁸⁵⁵ by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser⁶⁹⁶, Thr⁶⁹⁷, Ser⁸⁵⁴, and Thr⁸⁵⁵ in rat caudal artery, whereas U46619 induced Thr⁶⁹⁷ and Thr⁸⁵⁵ phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.     

Distinct Sarcomeric Substrates Are Responsible for Protein Kinase D-mediated Regulation of Cardiac Myofilament Ca2+ Sensitivity and Cross-bridge Cycling

Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser²²/Ser²³, reduces myofilament Ca²⁺ sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser²²/Ser²³ remains to be established. To determine the role of cTnI phosphorylation at Ser²²/Ser²³ in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser²²/Ser²³ are substituted by nonphosphorylatable Ala (cTnI-Ala₂). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser²²/Ser²³ and decreased the Ca²⁺ sensitivity of force. In contrast, PKD had no effect on the Ca²⁺ sensitivity of force in myocardium from cTnI-Ala₂ mice, in which Ser²²/Ser²³ were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala₂ mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser²⁷³, Ser²⁸², and Ser³⁰², and revealed that PKD phosphorylates only Ser³⁰². Furthermore, PKD phosphorylated Ser³⁰² selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala₂ mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca²⁺ sensitivity through cTnI phosphorylation at Ser²²/Ser²³ but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser³⁰², which may mediate the latter effect.     

Identification of phosphorylation sites in the COOH‐terminal tail of the μ‐opioid receptor

Phosphorylation is considered a key event in the signalling and regulation of the μ opioid receptor (MOPr). Here, we used mass spectroscopy to determine the phosphorylation status of the C‐terminal tail of the rat MOPr expressed in human embryonic kidney 293 (HEK‐293) cells. Under basal conditions, MOPr is phosphorylated on Ser³⁶³ and Thr³⁷⁰, while in the presence of morphine or [D‐Ala2, NMe‐Phe4, Gly‐ol5]‐enkephalin (DAMGO), the COOH terminus is phosphorylated at three additional residues, Ser³⁵⁶, Thr³⁵⁷ and Ser³⁷⁵. Using N‐terminal glutathione S transferase (GST) fusion proteins of the cytoplasmic, C‐terminal tail of MOPr and point mutations of the same, we show that, in vitro, purified G protein‐coupled receptor kinase 2 (GRK2) phosphorylates Ser³⁷⁵, protein kinase C (PKC) phosphorylates Ser³⁶³, while CaMKII phosphorylates Thr³⁷⁰. Phosphorylation of the GST fusion protein of the C‐terminal tail of MOPr enhanced its ability to bind arrestin‐2 and ‐3. Hence, our study identifies both the basal and agonist‐stimulated phospho‐acceptor sites in the C‐terminal tail of MOPr, and suggests that the receptor is subject to phosphorylation and hence regulation by multiple protein kinases.     

Phosphorylation of phospholamban in ischemia-reperfusion injury: Functional role of Thr17 residue

Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser¹⁶ by PKA and/or at Thr¹⁷ by CaMKII increases the affinity of the SR Ca²⁺ pump for Ca²⁺. PLB is therefore, a critical regulator of SR function, myocardial relaxation and myocardial contractility. The present study was undertaken to examine the status of PLB phosphorylation after ischemia and reperfusion and to provide evidence about the possible role of the phosphorylation of Thr¹⁷ PLB residue on the recovery of contractility and relaxation after a period of ischemia. Experiments were performed in Langendorff perfused hearts from Wistar rats. Hearts were submitted to a protocol of global normothermic ischemia and reperfusion. The results showed that (1) the phosphorylation of Ser¹⁶ and Thr¹⁷ residues of PLB increased at the end of the ischemia and the onset of reperfusion, respectively. The increase in Thr¹⁷ phosphorylation was associated with a recovery of relaxation to preischemic values. This recovery occurred in spite of the fact that contractility was depressed. (2) The reperfusion-induced increase in Thr¹⁷ phosphorylation was dependent on Ca²⁺ entry to the cardiac cell. This Ca²⁺ influx would mainly occur by the coupled activation of the Na⁺/H⁺ exchanger and the Na⁺/Ca²⁺ exchanger working in the reverse mode, since phosphorylation of Thr¹⁷ was decreased by inhibition of these exchangers and not affected by blockade of the L-type Ca²⁺ channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased Thr¹⁷ phosphorylation. This decrease was associated with an impairment of myocardial relaxation. The present study suggests that the phosphorylation of Thr¹⁷ of PLB upon reflow, may favor the full recovery of relaxation after ischemia. (Mol Cell Biochem 263: 131–136, 2004)     

Methylglyoxal Activates the Target of Rapamycin Complex 2-Protein Kinase C Signaling Pathway in Saccharomyces cerevisiae

Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We show here that methylglyoxal activates the Pkc1-Mpk1 mitogen-activated protein (MAP) kinase cascade in a target of rapamycin complex 2 (TORC2)-dependent manner in the budding yeast Saccharomyces cerevisiae. We demonstrate that TORC2 phosphorylates Pkc1 at Thr¹¹²⁵ and Ser¹¹⁴³. Methylglyoxal enhanced the phosphorylation of Pkc1 at Ser¹¹⁴³, which transmitted the signal to the downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of Pkc1^T1125 affected the phosphorylation of Pkc1 at Ser¹¹⁴³, in addition to its protein levels. Methylglyoxal activated mammalian TORC2 signaling, which, in turn, phosphorylated Akt at Ser⁴⁷³. Our results suggest that methylglyoxal is a conserved initiator of TORC2 signaling among eukaryotes.     

Leptin and insulin induce mutual resistance for nitric oxide synthase III activation in adipocytes

Obesity‐induced hyperleptinemia is frequently associated with insulin resistance suggesting a crosstalk between leptin and insulin signaling pathways. Our aim was to determine whether insulin and leptin together interfere on NOS activation in adipocytes. We examined insulin and leptin‐induced nitric oxide synthase (NOS) activity, protein amount and NOS III phosphorylation at Ser¹¹⁷⁹ in isolated epididymal adipocytes from rat, in the presence or not of inhibitors of kinases implicated in insulin or leptin signaling pathways. Insulin or leptin induced NOS III phosphorylation at Ser¹¹⁷⁹ leading to increased NO production in rat adipocytes, in agreement with our previous observations. When insulin and leptin at a concentration found in obese rats (10 ng/ml) were combined, NOS activity was not increased, suggesting a negative crosstalk between insulin and leptin signaling mechanisms. Chemical inhibitors of kinases implicated in signaling pathways of insulin, such as PI‐3 kinase, or of leptin, such as JAK‐2, did not prevent this negative interaction. When leptin signaling was blocked by PKA inhibitors, insulin‐induced NOS activity and NOS III phosphorylation at Ser¹¹⁷⁹ was observed. In the presence of leptin and insulin, (i) IRS‐1 was phosphorylated on Ser³⁰⁷ and this effect was prevented by PKA inhibitors, (ii) JAK‐2 was dephosphorylated, an effect prevented by SHP‐1 inhibitor. A mutual resistance occurs with leptin and insulin. Leptin phosphorylates IRS‐1 to induce insulin resistance while insulin dephosphorylates JAK‐2 to favor leptin resistance. This interference between insulin and leptin signaling could play a crucial role in insulin‐ and leptin‐resistance correlated with obesity. J. Cell. Biochem. 108: 982–988, 2009. © 2009 Wiley‐Liss, Inc.     

Protein Kinase D Interacts with Neuronal Nitric Oxide Synthase and Phosphorylates the Activatory Residue Serine1412

Neuronal Nitric Oxide Synthase (nNOS) is the biosynthetic enzyme responsible for nitric oxide (·NO) production in muscles and in the nervous system. This constitutive enzyme, unlike its endothelial and inducible counterparts, presents an N-terminal PDZ domain known to display a preference for PDZ-binding motifs bearing acidic residues at -2 position. In a previous work, we discovered that the C-terminal end of two members of protein kinase D family (PKD1 and PKD2) constitutes a PDZ-ligand. PKD1 has been shown to regulate multiple cellular processes and, when activated, becomes autophosphorylated at Ser⁹¹⁶, a residue located at -2 position of its PDZ-binding motif. Since nNOS and PKD are spatially enriched in postsynaptic densities and dendrites, the main objective of our study was to determine whether PKD1 activation could result in a direct interaction with nNOS through their respective PDZ-ligand and PDZ domain, and to analyze the functional consequences of this interaction. Herein we demonstrate that PKD1 associates with nNOS in neurons and in transfected cells, and that kinase activation enhances PKD1-nNOS co-immunoprecipitation and subcellular colocalization. However, transfection of mammalian cells with PKD1 mutants and yeast two hybrid assays showed that the association of these two enzymes does not depend on PKD1 PDZ-ligand but its pleckstrin homology domain. Furthermore, this domain was able to pull-down nNOS from brain extracts and bind to purified nNOS, indicating that it mediates a direct PKD1-nNOS interaction. In addition, using mass spectrometry we demonstrate that PKD1 specifically phosphorylates nNOS in the activatory residue Ser¹⁴¹², and that this phosphorylation increases nNOS activity and ·NO production in living cells. In conclusion, these novel findings reveal a crucial role of PKD1 in the regulation of nNOS activation and synthesis of ·NO, a mediator involved in physiological neuronal signaling or neurotoxicity under pathological conditions such as ischemic stroke or neurodegeneration.     

Interdependent Phosphorylation within the Kinase Domain T-loop Regulates CHK2 Activity

Chk2 is a critical regulator of the cellular DNA damage repair response. Activation of Chk2 in response to IR-induced damage is initiated by phosphorylation of the Chk2 SQ/TQ cluster domain at Ser¹⁹, Ser³³, Ser³⁵, and Thr⁶⁸. This precedes autophosphorylation of Thr³⁸³/Thr³⁸⁷ in the T-loop region of the kinase domain an event that is a prerequisite for efficient kinase activity. We conducted an in-depth analysis of phosphorylation within the T-loop region (residues 366–406). We report four novel phosphorylation sites at Ser³⁷², Thr³⁷⁸, Thr³⁸⁹, and Tyr³⁹⁰. Substitution mutation Y390F was defective for kinase function. The substitution mutation T378A ablated the IR induction of kinase activity. Interestingly, the substitution mutation T389A demonstrated a 6-fold increase in kinase activity when compared with wild-type Chk2. In addition, phosphorylation at Thr³⁸⁹ was a prerequisite to phosphorylation at Thr³⁸⁷ but not at Thr³⁸³. Quantitative mass spectrometry analysis revealed IR-induced phosphorylation and subcellular distribution of Chk2 phosphorylated species. We observed IR-induced increase in phosphorylation at Ser³⁷⁹, Thr³⁸⁹, and Thr³⁸³/Thr³⁸⁹. Phosphorylation at Tyr³⁹⁰ was dramatically reduced following IR. Exposure to IR was also associated with changes in the ratio of chromatin/nuclear localization. IR-induced increase in chromatin localization was associated with phosphorylation at Thr³⁷², Thr³⁷⁹, Thr³⁸³, Thr³⁸⁹, Thr³⁸³/Thr³⁸⁷, and Thr³⁸³/Thr³⁸⁹. Chk2 hyper-phosphorylated species at Thr³⁸³/Thr³⁸⁷/Thr³⁸⁹ and Thr³⁸³/Thr³⁸⁷/Thr³⁸⁹/Tyr³⁹⁰ relocalized from almost exclusively chromatin to predominately nuclear expression, suggesting a role for phosphorylation in regulation of chromatin targeting and egress. The differential impact of T-loop phosphorylation on Chk2 ubiquitylation suggests a co-dependence of these modifications. The results demonstrate that a complex interdependent network of phosphorylation events within the T-loop exchange region regulates dimerization/autophosphorylation, kinase activation, and chromatin targeting/egress of Chk2.     

New Hierarchical Phosphorylation Pathway of the Translational Repressor eIF4E-binding Protein 1 (4E-BP1) in Ischemia-Reperfusion Stress

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr⁶⁹-phosphorylated alone, Thr⁶⁹- and Thr³⁶/Thr⁴⁵-phosphorylated, all these plus Ser⁶⁴ phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr³⁶/Thr⁴⁵ phosphorylation alone was detected without Thr⁶⁹ phosphorylation, and neither was Ser⁶⁴ phosphorylation without Thr³⁶/Thr⁴⁵/Thr⁶⁹ phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr⁶⁹, Thr³⁶/Thr⁴⁵, and Ser⁶⁴ residues, with 4E-BP1 remaining phosphorylated at Thr⁶⁹ alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr³⁶/Thr⁴⁵ and Ser⁶⁴, in addition to Thr⁶⁹. Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr⁶⁹ is phosphorylated first followed by Thr³⁶/Thr⁴⁵ phosphorylation, and Ser⁶⁴ is phosphorylated last. Thr⁶⁹ phosphorylation alone allows binding to eIF4E, and subsequent Thr³⁶/Thr⁴⁵ phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.     

Phospholamban phosphorylation in ischemia-reperfused heart. Effect of pacing during ischemia and response to a β-adrenergic challenge

The status of phospholamban (PLB) phosphorylation in the ischemia-reperfused hearts remains controversial. Although a decrease in the phosphorylation of both PLB residues (Ser¹⁶, PKA site, and Thr¹⁷, CaMKII site) was previously reported, experiments from our laboratory failed to detect this decrease. In an attempt to elucidate the cause for this discrepancy, experiments were performed in Langendorff-perfused rat hearts with two main goals: (1) To determine whether keeping pacing during ischemia, a protocol followed in other ischemia-reperfusion models, decreases the phosphorylation of PLB residues, below pre-ischemic values; (2) To investigate whether a maximal -adrenergic challenge allows to detect a decrease in the ability of PLB to be phosphorylated in ischemia-reperfused hearts. Hearts were submitted to a global ischemia/reperfusion protocol (20/30 min) with (P) or without (NP) pacing during ischemia, and phosphorylation of PLB residues was assessed by immunodetection. The recovery of contractility upon reperfusion was lower in P vs. NP hearts. Ser¹⁶ of PLB, was phosphorylated at the end of ischemia in NP hearts. This increase appeared earlier in P hearts and was significantly diminished by catecholamine depletion and -blockade. Thr¹⁷ site was phosphorylated at the beginning of ischemia and the onset of reperfusion. The ischemia-induced phosphorylation of Thr¹⁷ was higher and more sustained in P vs. NP hearts, and inhibited by the calcium channel blocker, nifedipine, whereas the reperfusion-induced increase in Thr¹⁷ phosphorylation was similar in P and NP hearts and was significantly diminished by the Na⁺/Ca²⁺ exchanger inhibitor KB-R7943. Phosphorylation of PLB residues did not decrease below basal levels at any time during ischemia and reperfusion. However, the phosphorylation, inotropic and lusitropic response to -adrenergic stimulation was significantly decreased both in P and NP hearts.     

Tianeptine potentiates AMPA receptors by activating CaMKII and PKA via the p38, p42/44 MAPK and JNK pathways

Impairments of cellular plasticity appear to underlie the pathophysiology of major depression. Recently, elevated levels of phosphorylated AMPA receptor were implicated in the antidepressant effect of various drugs. Here, we investigated the effects of an antidepressant, Tianeptine, on synaptic function and GluA1 phosphorylation using murine hippocampal slices and in vivo single-unit recordings. Tianeptine, but not imipramine, increased AMPA receptor-mediated neuronal responses both in vitro and in vivo, in a staurosporine-sensitive manner. Paired-pulse ratio was unaltered by Tianeptine, suggesting a postsynaptic site of action. Tianeptine, 10 μM, enhanced the GluA1-dependent initial phase of LTP, whereas 100 μM impaired the latter phases, indicating a critical role of GluA1 subunit phosphorylation in the excitation. Tianeptine rapidly increased the phosphorylation level of Ser⁸³¹-GluA1 and Ser⁸⁴⁵-GluA1. Using H-89 and KN-93, we show that the activation of both PKA and CaMKII is critical in the effect of Tianeptine on AMPA responses. Moreover, the phosphorylation states of Ser^217/221-MEK and Thr¹⁸³/Tyr¹⁸⁵-p42MAPK were increased by Tianeptine and specific kinase blockers of the MAPK pathways (PD 98095, SB 203580 and SP600125) prevented the effects of Tianeptine. Overall these data suggest that Tianeptine potentiates several signaling cascades associated with synaptic plasticity and provide further evidence that a major mechanism of action for Tianeptine is to act as an enhancer of glutamate neurotransmission via AMPA receptors.     

Ischemia induces phospholamban dephosphorylation via activation of calcineurin, PKC-α, and protein phosphatase 1, thereby inducing calcium overload in reperfusion

Cardiac sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA2a) promotes Ca²⁺ uptake in the SR. Dephosphorylated phospholamban (PLB) inhibits SERCA2a activity. We found a distinct dephosphorylation of PLB at Thr¹⁷ and Ser¹⁶ after 20-30 min of ischemia produced by coronary artery occlusion in rats. The aim of the study was to investigate how PLB is dephosphorylated in ischemia and to determine whether PLB dephosphorylation causes myocardial hypercontraction and calpain activation through Ca²⁺ overload in reperfusion. Protein inhibitor-1 (I-1) specifically inhibits protein phosphatase 1 (PP1), the predominant PLB phosphatase in heart. A Ca²⁺-dependent phosphatase calcineurin may also induce PLB dephosphorylation. Ischemia for 30 min induced PKC-α translocation, resulting in inactivation of I-1 through PKC-α-dependent phosphorylation at Ser⁶⁷. The PP1 activation following I-1 inactivation was thought to induce PLB dephosphorylation in ischemia. Ischemia for 30 min activated calcineurin, and pre-treatment with a calcineurin inhibitor, cyclosporine A (CsA), inhibited PKC-α translocation, I-1 phosphorylation at Ser⁶⁷, and PLB dephosphorylation in ischemia. Reperfusion for 5 min following 30 min of ischemia induced spreading of contraction bands (CBs) and proteolysis of fodrin by calpain. Both CsA and an anti-PLB antibody that inhibits binding of PLB to SERCA2a reduced the CB area and fodrin breakdown after reperfusion. These results reveal a novel pathway via which ischemia induces calcineurin-dependent activation of PKC-α, inactivation of I-1 through PKC-α-dependent phosphorylation at Ser⁶⁷, and PP1-dependent PLB dephosphorylation. The pathway contributes to the spreading of CBs and calpain activation through Ca²⁺ overload in early reperfusion.     

TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser⁴⁷³ in the hydrophobic motif, along with Thr³⁰⁸ in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr³⁰⁸, but the kinase(s) responsible for phosphorylating Akt at Ser⁴⁷³ (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser⁴⁷³ phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser⁴⁷³ in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.     

Poster Sessions CP13: Hypoxia,Ischemia and Oxidative Stress. Changes of brain nitric oxide production in experimental cerebral ischemia

In order to study the role of nitric oxide (NO) in ischemic brain injury. Global cerebral ischemia was established in SD rats by modified Pulsinelli's method. The activities of constitutive nitric oxide synthase (cNOS), inducible NOS (iNOS), neuronal NOS (nNOS), nitrite (NO₂) and cyclic GMP in cerebral cortex, hippocampus, striatum and cerebellum at different time intervals were measured by radioimmunoassy, NADPH‐d histochemistry and fluorometry methods. The results showed that the activities of cNOS increased at 5 min in four regions and decreased in cortex, hippocampus and striatum at 60 min, in cerebellum at 15 min iNOS increased in cortex and striatum at 15 min, in hippocampus and cerebellum at 10 min, and persisted to 60 min. The expression of nNOS increased after 5 min ischemia in cortex, striatum and hippocampus, and return to normal at 30–60 min. The NO₂ and cGMP also increased after 5–15 min ischemia and returned to normal after 30–60 min ischemia. These results indicated that the NO participated in the pathogenesis of cerebral ischemia injury and different types of NOS play different role in the cerebral ischemia injuries. Selected specific NOS inhibitors to decreased the excessive production of NO at early stage may help to decrease the ischemic injury.     

Regulation of mTOR Complex 1 (mTORC1) by Raptor Ser863 and Multisite Phosphorylation

The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTOR-interacting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (cluster 1, Ser⁶⁹⁶/Thr⁷⁰⁶ and cluster 2, Ser⁸⁵⁵/Ser⁸⁵⁹/Ser⁸⁶³/Ser⁸⁷⁷) using tandem mass spectrometry and generated phosphospecific antibodies for each of these sites. Here we focus primarily although not exclusively on raptor Ser⁸⁶³ phosphorylation. We report that insulin promotes mTORC1-associated phosphorylation of raptor Ser⁸⁶³ via the canonical PI3K/TSC/Rheb pathway in a rapamycin-sensitive manner. mTORC1 activation by other stimuli (e.g. amino acids, epidermal growth factor/MAPK signaling, and cellular energy) also promote raptor Ser⁸⁶³ phosphorylation. Rheb overexpression increases phosphorylation on raptor Ser⁸⁶³ as well as on the five other identified sites (e.g. Ser⁸⁵⁹, Ser⁸⁵⁵, Ser⁸⁷⁷, Ser⁶⁹⁶, and Thr⁷⁰⁶). Strikingly, raptor Ser⁸⁶³ phosphorylation is absolutely required for raptor Ser⁸⁵⁹ and Ser⁸⁵⁵ phosphorylation. These data suggest that mTORC1 activation leads to raptor multisite phosphorylation and that raptor Ser⁸⁶³ phosphorylation functions as a master biochemical switch that modulates hierarchical raptor phosphorylation (e.g. on Ser⁸⁵⁹ and Ser⁸⁵⁵). Importantly, mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward the substrate 4EBP1, with a multisite raptor 6A mutant more strongly defective that single-site raptor S863A. Taken together, these data suggest that complex raptor phosphorylation functions as a biochemical rheostat that modulates mTORC1 signaling in accordance with environmental cues.     

The developmental stage and cell type dependent phosphorylation of eNOS in murine enteric mucosa and myenteric plexus

In order to clarify the developmental regulation of the eNOS activity in intestine by phosphorylation, we examined the immunohistochemical localizations of the eNOS phosphorylation sites at Ser¹¹⁷⁷, Ser¹¹⁶ and at Thr⁴⁹⁵ in cells of the mouse enteric mucosa and myenteric plexus at E13.5, E14.5, E16.5, E18.5, E20.5 and P3. In addition, in cells of the E16.5 stage the protein levels of eNOS and the phosphorylation sites of eNOS at Ser¹¹⁷⁷, Ser¹¹⁶ and at Thr⁴⁹⁵ were investigated by immunoblot. From E14.5 to P3, phosphorylation residues of eNOS at Ser¹¹⁷⁷ and at Ser¹¹⁶ were detected with different staining intensities in the enteric mucosa epithelium. In ganglion cells of the myenteric plexus Ser¹¹⁶ was identified at E18.5 to P3. The absence of phosphorylated Thr⁴⁹⁵ in cells of intestine during all developmental stages, was confirmed by immunoblot at E16.5. The immunoblot levels of eNOS and eNOS phosphorylated at Ser¹¹⁷⁷ and at Ser¹¹⁶ were comparable with the immunohistochemical results of E16.5 mouse intestine. It was concluded that development of epithelial cells of the enteric mucosa may be modulated by phosphorylation of eNOS at Ser¹¹⁷⁷ and at Ser¹¹⁶. The phosphorylation of eNOS in cells of the myenteric plexus is modulated at Ser¹¹⁶. These data suggest that there is a developmental stage and cell type dependent phosphorylation of eNOS.