Exercise training improves cardiac performance in diabetes: in vivo demonstration with quantitative cine-MRI analyses.

Diabetic cardiomyopathy is a distinct myocardial complication of the catabolic state of untreated insulin-dependent diabetes mellitus in the streptozotocin-induced diabetic rat. Exercise training has long been utilized as an effective adjunct to pharmacotherapy in the management of the diabetic heart. However, the in vivo functional benefit(s) of the training programs on cardiac cycle events in diabetes are poorly understood. In this study, we used three groups of Sprague-Dawley rats (sedentary control, sedentary diabetic, and exercised diabetic) to assess the effects of endurance training on the left ventricular (LV) cardiac cycle events in diabetes. At the end of 9 wk of exercise training, noninvasive cardiac functional evaluation was performed by using high-resolution magnetic resonance imaging (9.4 T). An ECG-gated cine imaging protocol was used to capture the LV cardiac cycle events through 10 equally incremented phases. The cardiac cycle phase volumetric profiles showed favorable functional changes in exercised diabetic group, including a prevention of decreased end-diastolic volume and attenuation of increased end-systolic volume that accompanies sedentary diabetes. The defects in LV systolic flow velocity, acceleration, and jerk associated with sedentary diabetes were restored toward control levels in the trained diabetic animals. This magnetic resonance imaging study confirms the prevailing evidence from earlier in vitro and in vivo invasive procedures that exercise training benefits cardiac function in this model of diabetic cardiomyopathy despite the extreme catabolic state of the animals.     

Inhibition of cyclooxygenase-2 improves cardiac function in myocardial infarction

Induction of cyclooxygenase-2 (COX-2) in ischemic myocardium is thought to increase the production of proinflammatory prostanoids and contribute significantly to the ischemic inflammation. Left ventricular myocardial infarction (MI) was created by ligating the left coronary artery in Lewis rats. Hemodynamic measurements at 4 weeks showed better cardiac function in the group treated with a selective COX-2 inhibitor (DFU; 5 mg/kg/day) for 2 weeks after induction of MI compared to the vehicle treated group. These results suggest that induction of COX-2 contributes to myocardial dysfunction, and that selective inhibition of COX-2 could constitute an important therapeutic target for the treatment of MI.     

alpha-Adrenergic response and myofilament activity in mouse hearts lacking PKC phosphorylation sites on cardiac TnI

Protein kinase C (PKC)-mediated phosphorylation of cardiac myofilament (MF) proteins has been shown to depress the actomyosin interaction and may be important during heart failure. Biochemical studies indicate that phosphorylation of Ser(43) and Ser(45) of cardiac troponin I (cTnI) plays a substantial role in the PKC-mediated depression. We studied intact and detergent-extracted papillary muscles from nontransgenic (NTG) and transgenic (TG) mouse hearts that express a mutant cTnI (Ser43Ala, Ser45Ala) that lacks specific PKC-dependent phosphorylation sites. Treatment of NTG papillary muscles with phenylephrine (PE) resulted in a transient increase and a subsequent 62% reduction in peak twitch force. TG muscles showed no transient increase and only a 45% reduction in force. There was a similar difference in maximum tension between NTG and TG fiber bundles that had been treated with a phorbol ester and had received subsequent detergent extraction. Although levels of cTnI phosphorylation correlated with these differences, the TG fibers also demonstrated a decrease in phosphorylation of cardiac troponin T. The PKC-specific inhibitor chelerythrine inhibited these responses. Our data provide evidence that specific PKC-mediated phosphorylation of Ser(43) and Ser(45) of cTnI plays an important role in regulating force development in the intact myocardium.     

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.     

In vivo and in vitro analysis of cardiac troponin I phosphorylation

Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during beta-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser(23)/Ser(24)) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation.     

Inhibition and stimulation of growth of Entamoeba histolytica in culture: association with PKC activity and protein phosphorylation

We studied the role of protein kinase C (PKC) and protein threonine phosphorylation in the inhibition and stimulation of growth of the protozoan parasite Entamoeba histolytica. PKC was activated after serum deprivation in E. histolytica and during this period proteins became threonine phosphorylated. Conversely, on serum stimulation of serum-deprived cells, PKC activation was rapidly reversed and the threonine phosphorylation of proteins quickly declined. Growth of E. histolytica was not affected by either PKC inhibitors H-7 and GF109203X or by down-regulation of PKC by Phorbol 12-Myristate 13-Acetate (PMA). Interestingly, very low doses of PMA which caused activation of PKC and were unable to down-regulate PKC after 48 h of culture, negatively influenced the growth of E. histolytica. Serine/threonine phosphatase inhibitors Okadaic acid and Calyculin A drastically inhibited growth of E. histolytica. In conclusion, the growth of E. histolytica is not adversely affected by PKC down-regulation. On the contrary, growth inhibition of E. histolytica is associated with activation of Ca(2+), Diacylglycerol (DAG)-dependent PKC, and threo nine phosphorylation of proteins.     

Inhibition of cyclooxygenase-2 improves cardiac function after myocardial infarction in the mouse

Cyclooxygenase (COX)-2 is expressed in the heart in animal models of ischemic injury. Recent studies have suggested that COX-2 products are involved in inflammatory cell infiltration and fibroblast proliferation in the heart. Using a mouse model, we questioned whether 1). myocardial infarction (MI) in vivo induces COX-2 expression chronically, and 2). COX-2 inhibition reduces collagen content and improves cardiac function in mice with MI. MI was produced by ligation of the left anterior descending coronary artery in mice. Two days later, mice were treated with 3 mg/kg NS-398, a selective COX-2 inhibitor, or vehicle in drinking water for 2 wk. After the treatment period, mice were subjected to two-dimensional M-mode echocardiography to determine cardiac function. Hearts were then analyzed for determination of infarct size, interstitial collagen content, brain natriuretic peptide (BNP) mRNA, myocyte cross-sectional area, and immunohistochemical staining for transforming growth factor (TGF)-beta and COX-2. COX-2 protein, detected by immunohistochemistry, was increased in MI versus sham hearts. MI resulted in increased left ventricular systolic and diastolic dimension and decreased ejection fraction, fractional shortening, and cardiac output. NS-398 treatment partly reversed these detrimental changes. Myocyte cross-sectional area, a measure of hypertrophy, was decreased by 30% in the NS-398 versus vehicle group, but there was no effect on BNP mRNA. The interstitial collagen fraction increased from 5.4 +/- 0.4% in sham hearts to 10.4 +/- 0.9% in MI hearts and was decreased to 7.9 +/- 0.6% in NS-398-treated hearts. A second COX-2 inhibitor, rofecoxib (MK-0966), also decreased myocyte cross-sectional area and interstitial collagen fraction. TGF-beta, a key regulator of collagen synthesis, was increased in MI hearts. NS-398 treatment reduced TGF-beta immunostaining by 40%. NS-398 treatment had no effect on infarct size. These results suggest that COX-2 products contribute to cardiac remodeling and functional deficits after MI. Thus selected inhibition of COX-2 may be a therapeutic target for reducing myocyte damage after MI.     

Arginine in burn injury improves cardiac performance and prevents bacterial translocation

Horton, Jureta W., Jean White, David Maass, and BillySanders. Arginine in burn injury improves cardiac performance andprevents bacterial translocation. J. Appl.Physiol. 84(2): 695-702, 1998.This studyexamined the effects of arginine supplement of fluid resuscitation fromburn injury on cardiac contractile performance and bacterialtranslocation after a third-degree burn comprising 43% of the totalbody surface area in adult rats. Before burn injury, rats wereinstrumented to measure blood pressure; after burn (or sham injury),paired groups of sham-burned and burned rats were given vehicle(saline), L-arginine,D-arginine, orN-methyl-L-arginine(300 mg/kg in 0.3 ml of saline 30 min, 6 h, and 23 h postburn) plusfluid resuscitation; sham-burned rats received drug only.Twenty-four hours after burn trauma, hemodynamics were measured; theanimals were then killed and randomly assigned to Langendorff heartstudies or to studies examining translocation of gut bacteria. Burnrats treated with vehicle, D-arginine, orN-methyl-L-argininehad well-defined cardiocirculatory responses that included hypotension,tachycardia, respiratory compensation for metabolic acidosis,hypocalcemia, cardiac contractile depression, and significant bacterialtranslocation. Compared with values measured in vehicle-treated burnrats, L-arginine given afterburn improved blood pressure, prevented tachycardia, reduced serumlactate levels, improved cardiac performance, and significantly reducedbacterial translocation, confirming that L-arginine administration afterburn injury provided significant cardiac and gastrointestinalprotection. Circulating neutrophil counts fell after burn trauma andserum glucagon levels rose, but these changes were not altered bypharmacological intervention. Our finding of significantly highercoronary perfusate guanosine 3,5-cyclic monophosphateconcentration inL-arginine-treated burn ratssuggests that the beneficial effects ofL-arginine were mediated bynitric oxide production.

     

Oral administration of sodium tungstate improves cardiac performance in streptozotocin-induced diabetic rats

Normalization of hyperglycemia and hyperlipidemia is an important objective in preventing diabetes-induced cardiac dysfunction. Our study investigated the effects of sodium tungstate on cardiac performance in streptozotocin-induced (STZ) diabetic rats based on its potential antidiabetic and antioxidant activity. Male Wistar rats were made STZ-diabetic and then treated with tungstate in their drinking water for 9 weeks. Body mass, food and fluid intake, plasma glucose, insulin, triglyceride, and free fatty acids levels were measured. At the termination of the study period, an oral glucose tolerance test (OGTT) was performed, and cardiac performance was evaluated using an isolated working heart apparatus. Tungstate-treated STZ-diabetic rats showed a significant reduction in fluid and food intake, plasma glucose, triglycerides, and free fatty acid levels, and improved tolerance to glucose in OGTT, owing to tungstate-mediated enhancement of insulin activity rather than increased insulin levels. Left ventricular pressure development, the rate of contraction (+dP/dT), and the rate of relaxation (-dP/dT) were significantly improved in tungstate-treated diabetic rats. Apart from a decreased rate of body mass gain, no other signs of toxicity or hypoglycemic episodes were observed in tungstate-treated rats. This study extends previous observations on the antidiabetic activities of tungstate, and also reports for the first time the salutary effects in preventing diabetic cardiomyopathy.     

In vivo therapy of myocardial infarction with mesenchymal stem cells modified with prostaglandin I synthase gene improves cardiac performance in mice

AimIntra-myocardial injection of adult bone marrow-derived stem cells (MSC) has recently been proposed as a therapy to repair damaged cardiomyocytes after acute myocardial infarction (AMI). PGI₂ has vasodilatation effects; however, the effects of combining both MSC and PGI₂ therapy on AMI have never been evaluated.Main methodsWe genetically enhanced prostaglandin I synthase (PGIS) gene expression in mouse mesenchymal stem cells (MSC) using lentiviral vector transduction (MSC_PGIS). Mice were subjected to an AMI model and injected (intra-myocardially) with either 5 × 10⁴ MSCs or MSC_PGIS before surgery. Fourteen days post AMI, mice were analyzed with echocardiography, immunohistochemistry, and apoptotic, and traditional tissue assays.Key findingsLenti-PGIS transduction did not change any characteristic of the MSCs. PGIS over-expressed MSCs secreted 6-keto-PGF1α in the culture medium and decreased free radical damage during hypoxia/re-oxygenation and H₂O₂ treatment. Furthermore, splenocyte proliferation was significantly suppressed with MSC_PGIS as compared with MSCs alone. Fourteen days post AMI, echocardiography showed more improvement in cardiac function of the MSC_PGIS group than the MSC alone group, sham-operated group, or artery ligation only group. The histology of MSC_PGIS treated hearts revealed MSCs in the infarcted region and decreased myocardial fibrosis/apoptosis with limited cardiac remodeling. Furthermore, the level of the vascular endothelial growth factor was elevated in the MSC_PGIS group as compared to the other three groups.SignificanceIn summary, our results provide both in vitro and in vivo evidence for the beneficial role of MSC_PGIS in limiting the process of detrimental cardiac remodeling in a mouse AMI model during early stages of the disease.     

Rapamycin-sensitive phosphorylation of PKC on a carboxy-terminal site by an atypical PKC complex.

BACKGROUND: The protein kinase C (PKC) family has been implicated in the control of many cellular functions. Although PKC isotypes are characterized by their allosteric activation, phosphorylation also plays a key role in controlling activity. In classical PKC isotypes, one of the three critical sites is a carboxy-terminal hydrophobic site also conserved in other AGC kinase subfamily members. Although this site is crucial to the control of this class of enzymes, the upstream kinase(s) has not been identified. RESULTS: A membrane-associated kinase activity that phosphorylates the hydrophobic site in PKCalpha was detected. This activity was suppressed when cells were pretreated with the immunosuppresant drug rapamycin or the phosphoinositide (Pl) 3-kinase inhibitor LY294002. These pretreatments also blocked specifically the serum-induced phosphorylation of the hydrophobic site in PKCdelta in vivo. The most highly purified hydrophobic site kinase preparations ( approximately 10,000-fold) reacted with antibodies to PKCzeta/iota. Consistent with this, rapamycin and LY294002 reduced the recovery of PKCzeta from the membrane fraction of transfected cells. An activated mutant of PKCzeta, but not wild-type PKCzeta, induced phosphorylation of the PKCdelta hydrophobic site in a rapamycin-independent manner, whereas a kinase-dead PKCzeta mutant suppressed this serum-induced phosphorylation. The immunopurified, activated mutant of PKCzeta could phosphorylate the PKCdelta hydrophobic site in vitro, whereas wild-type PKCzeta could not. CONCLUSIONS: PKCzeta is identified as a component of the upstream kinase responsible for the phosphorylation of the PKCdelta hydrophobic site in vitro and in vivo. PKCzeta can therefore control the phosphorylation of this PKCdelta site, antagonizing a rapamycin-sensitive pathway.     

PKCβ-dependent phosphorylation of the glycine transporter 1

The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [³²P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23–40%-inhibition on V_max was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKCα/β inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation.     

Regulation of membrane-bound PKC in adult cardiac ventricular myocytes

Activation of protein kinase C (PKC) is thought to involve translocation to the particulate fraction. The present study demonstrates a membrane-associated, inactive pool of PKC in adult rat ventricular myocytes. Membranes were isolated from stimulated (phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1)) or control myocytes and PKC activity determined in the absence (active PKC) or presence (total PKC) of PMA. An inactive, PMA-responsive, pool of PKC was detected. In intact myocytes, PMA or ET-1 induced a translocation of PKC epsilon from the cytosol into the particulate fraction. In contrast, ET-1 decreased both total and active PKC in the membranes: this decrease was associated with a loss of PKC epsilon immunoreactivity. PMA increased the amount of membrane-associated, inactive PKC. Our results demonstrate the presence of a membrane-associated pool of PKC in cardiac myocytes that is differentially modulated by ET-1 or PMA.     

PKC theta ablation improves healing in a mouse model of muscular dystrophy

Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, limiting muscle regeneration and resulting in fibrotic and fatty tissue replacement of muscle, which exacerbates the wasting process in dystrophic muscles. Limiting immune response is thus one of the therapeutic options to improve healing, as well as to improve the efficacy of gene- or cell-mediated strategies to restore dystrophin expression. Protein kinase C θ (PKCθ) is a member of the PKCs family highly expressed in both immune cells and skeletal muscle; given its crucial role in adaptive, but also innate, immunity, it is being proposed as a valuable pharmacological target for immune disorders. In our study we asked whether targeting PKCθ could represent a valuable approach to efficiently prevent inflammatory response and disease progression in a mouse model of muscular dystrophy. We generated the bi-genetic mouse model mdx/θ(-/-), where PKCθ expression is lacking in mdx mice, the mouse model of Duchenne muscular dystrophy. We found that muscle wasting in mdx/θ(-/-) mice was greatly prevented, while muscle regeneration, maintenance and performance was significantly improved, as compared to mdx mice. This phenotype was associated to reduction in inflammatory infiltrate, pro-inflammatory gene expression and pro-fibrotic markers activity, as compared to mdx mice. Moreover, BM transplantation experiments demonstrated that the phenotype observed was primarily dependent on lack of PKCθ expression in hematopoietic cells.These results demonstrate a hitherto unrecognized role of immune-cell intrinsic PKCθ activity in the development of DMD. Although the immune cell population(s) involved remain unidentified, our findings reveal that PKCθ can be proposed as a new pharmacological target to counteract the disease, as well as to improve the efficacy of gene- or cell- therapy approaches.     

Isolation of cardiac myofibrils and myosin light chains with in vivo levels of light chain phosphorylation

Conditions are described for the preparation of functional myofibrils and myosin light chains from freeze-clamped beating hearts with the state of light chain phosphorylation chemically ‘frozen’ during the extraction procedure. Myofibrils were shown to be functionally intact by measurement of Ca²⁺ binding and ATPase activity. Highly purified cardiac myosin light chains could be routinely isolated from myofibrillar preparations using ethanol fractionation together with ion-exchange chromotography. Analysis of light chains for covalent phosphate indicated that basal levels of phosphorylation of the 18?20 000 dalton light chain of myosin in rabbit hearts beating in situ or in a perfusion apparatus were 0.3–0.4 mol/mol. Covalent phosphate content of the light chain fraction did not change during perfusion of hearts with 10 μM epinephrine.     

Isolation of cardiac myofibrils and myosin light chains with in vivo levels of light chain phosphorylation.

Conditions are described for the preparation of functional myofibrils and myosin light chains from freeze-clamped beating hearts with the state of light chain phosphorylation chemically 'frozen' during the extraction procedure. Myofibrils were shown to be functionally intact by measurement of Ca2+ binding and ATPase activity. Highly purified cardiac myosin light chains could be routinely isolated from myofibrillar preparations using ethanol fractionation together with ion-exchange chromatography. Analysis of light chains for covalent phosphate indicated that basal levels of phosphorylation of the 18--20 000 dalton light chain of myosin in rabbit hearts beating in situ or in a perfusion apparatus were 0.3--0.4 mol/mol. Covalent phosphate content of the light chain fraction did not change during perfusion of hearts with 10 microM epinephrine.     

Inhibition of protein phosphorylation by chloroquine

The rapid phase of fructose-1,6-bisphosphatase (FBPase) inactivation following glucose addition to starved yeast cells [reported previously] is inhibited on addition of 10 mM chloroquine (CQ) at about pH 8. This inhibition of inactivation was shown to be due to the prevention of phosphorylation of the enzyme. CQ was also found to inhibit general protein phosphorylation in the yeast cells. Glycolysis, as observed by changes in intracellular glucose-6-phosphate and extracellular glucose and ethanol concentrations, was shown to be significantly inhibited in cells treated with CQ. Similarly, a decrease in ATP concentrations was observed. However, during the early stages of phosphorylation of FBPase, levels of ATP were similar in cells containing CQ as in those without CQ. Thus, decrease in ATP levels is not thought to be significantly responsible for the inhibition of protein phosphorylation. However, the phosphorylating activity of cyclic AMP-dependent protein kinases is inhibited in vitro by relatively low concentrations of CQ. Thus, prevention of protein phosphorylation by CQ is believed to be due to inhibition of protein kinases in yeast cells.Abbreviations FBPase fructose-1,6-bisphosphatase - CQ chloroquine - SDS sodium dodecyl sulfate - G6P glucose-6-phosphate - TCA trichloroacetic acid     

Stimulus-induced phosphorylation of PKC theta at the C-terminal hydrophobic-motif in human T lymphocytes

Protein kinase C (PKC) is a family of serine/threonine kinases whose activity is controlled, in part, by phosphorylation on three conserved residues that are located on the catalytic domain of the enzyme, known as the activation-loop, the turn-motif, and the C-terminal hydrophobic-motif sites. Using a panel of phospho-specific antibodies, we have determined that PKC beta(I) and delta are constitutively phosphorylated on all three sites in unstimulated and activated T cells. Although PKC theta is constitutively phosphorylated at the activation-loop and turn-motif sites in T cells, PMA or anti-CD3/CD28 stimulation results in an increase in phosphorylation at the hydrophobic-motif (Ser695), an event that coincides with translocation of the enzyme from the cytosol/cytoskeleton to the membrane. Studies on the stimulus-induced phosphorylation of PKC theta demonstrate that an upstream kinase activity involving a conventional PKC isoform(s) and the PI3-kinase pathway, rather than autophosphorylation or the rapamycin-sensitive mTOR pathway, regulates this site in T lymphocytes. However, hydrophobic-motif phosphorylation does not appear to control membrane translocation, suggesting that this site may control other aspects of PKC theta signalling.