5'AMP activated protein kinase expression in human skeletal muscle: effects of strength training and type 2 diabetes

Strength training enhances insulin sensitivity and represents an alternative to endurance training for patients with type 2 diabetes (T2DM). The 5'AMP-activated protein kinase (AMPK) may mediate adaptations in skeletal muscle in response to exercise training; however, little is known about adaptations within the AMPK system itself. We investigated the effect of strength training and T2DM on the isoform expression and the heterotrimeric composition of the AMPK in human skeletal muscle. Ten patients with T2DM and seven healthy subjects strength trained (T) one leg for 6 weeks, while the other leg remained untrained (UT). Muscle biopsies were obtained before and after the training period. Basal AMPK activity and protein/mRNA expression of both catalytic (α1 and α2) and regulatory (β1, β2, γ1, γ2a, γ2b and γ3) AMPK isoforms were independent of T2DM, whereas the protein content of α1 (+16%), β2 (+14%) and γ1 (+29%) was higher and the γ3 content was lower (−48%) in trained compared with untrained muscle (all P < 0.01). The majority of α protein co-immunoprecipitated with β2 and α2/β2 accounted for the majority of these complexes. γ3 was only associated with α2 and β2 subunits, and accounted for ∼20% of all α2/β2 complexes. The remaining α2/β2 and the α1/β2 complexes were associated with γ1. The trimer composition was unaffected by T2DM, whereas training induced a shift from γ3- to γ1-containing trimers. The data question muscular AMPK as a primary cause of T2DM whereas the maintained function in patients with T2DM makes muscular AMPK an obvious therapeutic target. In human skeletal muscle only three of 12 possible AMPK trimer combinations exist, and the expression of the subunit isoforms is susceptible to moderate strength training, which may influence metabolism and improve energy homeostasis in trained muscle.     

Proton modulation of recombinant GABAA receptors: influence of GABA concentration and the β subunit TM2–TM3 domain

Regulation of GABA_A receptors by extracellular pH exhibits a dependence on the receptor subunit composition. To date, the molecular mechanism responsible for the modulation of GABA_A receptors at alkaline pH has remained elusive. We report here that the GABA-activated current can be potentiated at pH 8.4 for both αβ and αβγ subunit-containing receptors, but only at GABA concentrations below the EC₄₀. Site-specific mutagenesis revealed that a single lysine residue, K279 in the β subunit TM2–TM3 linker, was critically important for alkaline pH to modulate the function of both α1β2 and α1β2γ2 receptors. The ability of low concentrations of GABA to reveal different pH titration profiles for GABA_A receptors was also examined at acidic pH. At pH 6.4, GABA activation of αβγ receptors was enhanced at low GABA concentrations. This effect was ablated by the mutation H267A in the β subunit. Decreasing the pH further to 5.4 inhibited GABA responses via αβγ receptors, whereas those responses recorded from αβ receptors were potentiated. Inserting homologous β subunit residues into the γ2 subunit to recreate, in αβγ receptors, the proton modulatory profile of αβ receptors, established that in the presence of β2^H267, the mutation γ2^T294K was necessary to potentiate the GABA response at pH 5.4. This residue, T294, is homologous to K279 in the β subunit and suggests that a lysine at this position is an important residue for mediating the allosteric effects of both acidic and alkaline pH changes, rather than forming a direct site for protonation within the GABA_A receptor.     

Interleukin-2 (IL-2) Receptor α, β and γ Subunit Expression as a Function of B-Cell Lineage Ontogeny: the Use of IL-2-PE664Glu to Characterize Internalization via IL-2 Receptor Subunits

Interleukin-2 (IL-2) is a pluripotent cytokine which plays a crucial role in the immune system response. Although the IL-2/IL-2 receptor (IL-2R) system has been well characterized in cells of the T lineage it is less known in B lymphocytes. The authors therefore studied the expression of the IL-2Rα, β and γ subunits in human B-cell lines at different stages of maturation, by the polymerase chain reaction technique. The authors found that the α and β subunits are expressed in the final stages of B-cell lineage maturation, whereas the γ subunit is constitutively expressed during B-lymphocyte differentiation. The results indicate that the IL-2/IL-2R system, most probably, does not have a role in the early stages of B-cell differentiation, but may be involved only in the final stages of B-cell lineage ontogeny. Moreover, the ability of the different forms of IL-2R to internalize the IL-2 ligand was investigated, using the chimeric protein IL-2-PE66^4Glu. Cell lines bearing the αγ, βγ and αβγ forms of IL-2R were inhibited by the chimeric protein, while those bearing the γ subunit alone did not respond to the chimera. Thus, internalization of IL-2 is most likely mediated via the αγ form of the IL-2R, as shown here for the first time, as well as through the βγ and αβγ IL-2R forms. However, IL-2 cannot be internalized through the IL-2R γ subunit alone.     

Higher skeletal muscle α2AMPK activation and lower energy charge and fat oxidation in men than in women during submaximal exercise

5'AMP-activated protein kinase (AMPK) is an energy sensor activated by perturbed cellular energy status such as during muscle contraction. Activated AMPK is thought to regulate several key metabolic pathways. We used sex comparison to investigate whether AMPK signalling in skeletal muscle regulates fat oxidation during exercise. Moderately trained women and men completed 90 min bicycle exercise at 60%     . Both AMPK Thr¹⁷² phosphorylation and α₂AMPK activity were increased by exercise in men (∼200%, P < 0.001) but not significantly in women. The sex difference in muscle AMPK activation with exercise was accompanied by an increase in muscle free AMP (∼164%, P < 0.01), free AMP/ATP ratio (159%, P < 0.05), and creatine (∼42%, P < 0.001) in men but not in women (NS), suggesting that lack of AMPK activation in women was due to better maintenance of muscle cellular energy balance compared with men. During exercise, fat oxidation per kg lean body mass was higher in women than in men ( P < 0.05). Regression analysis revealed that a higher proportion of type 1 muscle fibres (∼23%, P < 0.01) and a higher capillarization (∼23%, P < 0.05) in women than in men could partly explain the sex difference in α₂AMPK activity ( r =−0.54, P < 0.05) and fat oxidation ( r = 0.64, P < 0.05) during exercise. On the other hand, fat oxidation appeared not to be regulated via AMPK. In conclusion, during prolonged submaximal exercise at 60%     , higher fat oxidation in women cannot be explained by higher AMPK signalling but is accompanied by improved muscle cellular energy balance in women probably due to sex specific muscle morphology.     

γδ T-Cell Precursor-Derived CD4− CD8−αβ T Cells Retain γδ Cell Function

We have previously shown that some of the DNαβ⁺ T cells arising in TcRα-chain transgenic mice are of γδ T cell origin, based on phenotypic data and on their status of TcR gene rearrangements. In the present report we investigated the impact of αβ TcR expression on the functional programme of the mature γδ precursor-derived DNαβ⁺ T cells. Our results demonstrate that both their proliferative capacity and their cytokine production profile are similar to that of γδ T cells. Furthermore, both transgenic DNαβ⁺ T cells and DNγδ⁺ T cells up-regulate CD8α expression after activation, but, in contrast to CD4⁺αβ T cells, are unable to induce proliferation of naive B cells. Thus, our results suggest that the effector functions of mature T cells are determined independently of the TcR isotype, probably at an early stage of differentiation, and thereby have important implications for current models of T-cell lineage commitment.     

Short-term exercise training in humans reduces AMPK signalling during prolonged exercise independent of muscle glycogen

We examined the effect of short-term exercise training on skeletal muscle AMP-activated protein kinase (AMPK) signalling and muscle metabolism during prolonged exercise in humans. Eight sedentary males completed 120 min of cycling at 66 ± 1%     , then exercise trained for 10 days, before repeating the exercise bout at the same absolute workload. Participants rested for 72 h before each trial while ingesting a high carbohydrate diet (HCHO). Exercise training significantly ( P < 0.05) attenuated exercise-induced increases in skeletal muscle free AMP: ATP ratio and glucose disposal and increased fat oxidation. Exercise training abolished the 9-fold increase in AMPK α2 activity observed during pretraining exercise. Since training increased muscle glycogen content by 93 ± 12% ( P < 0.01), we conducted a second experiment in seven sedentary male participants where muscle glycogen content was essentially matched pre- and post-training by exercise and a low CHO diet (LCHO; post-training muscle glycogen 52 ± 7% less than in HCHO, P < 0.001). Despite the difference in muscle glycogen levels in the two studies we obtained very similar results. In both studies the increase in ACCβ Ser²²¹ phosphorylation was reduced during exercise after training. In conclusion, there is little activation of AMPK signalling during prolonged exercise following short-term exercise training suggesting that other factors are important in the regulation of glucose disposal and fat oxidation under these circumstances. It appears that muscle glycogen is not an important regulator of AMPK activation during exercise in humans when exercise is begun with normal or high muscle glycogen levels.     

Differential dissociation of G protein heterotrimers

Signalling by heterotrimeric G proteins is often isoform-specific, meaning certain effectors are regulated exclusively by one family of heterotrimers. For example, in excitable cells inwardly rectifying potassium (GIRK) channels are activated by Gβγ dimers derived specifically from G_i/o heterotrimers. Since all active heterotrimers are thought to dissociate and release free Gβγ dimers, it is unclear why these channels respond primarily to dimers released by G_i/o heterotrimers. We reconstituted GIRK channel activation in cells where we could quantify heterotrimer expression at the plasma membrane, GIRK channel activation, and heterotrimer dissociation. We find that G_oA heterotrimers are more effective activators of GIRK channels than G_s heterotrimers when comparable amounts of each are available. We also find that active G_oA heterotrimers dissociate more readily than active G_s heterotrimers. Differential dissociation may thus provide a simple explanation for Gα-specific activation of GIRK channels and other Gβγ-sensitive effectors.     

Molecular determinants of glycine receptor αβ subunit sensitivities to Zn2+-mediated inhibition

Glycine receptors exhibit a biphasic sensitivity profile in response to Zn²⁺-mediated modulation, with low Zn²⁺ concentrations potentiating (< 10 μ m ), and higher Zn²⁺ concentrations inhibiting submaximal responses to glycine. Here, a substantial 30-fold increase in sensitivity to Zn²⁺-mediated inhibition was apparent for the homomeric glycine receptor (GlyR) α1 subunit compared to either GlyR α2 or α3 subtypes. Swapping the divergent histidine (H107) residue in GlyR α1, which together with the conserved H109 forms part of an intersubunit Zn²⁺-binding site, for the equivalent asparagine residue present in GlyR α2 and α3, reversed this phenotype. Co-expression of heteromeric GlyR α1 or α2 with the ancillary β subunit yielded receptors that maintained their distinctive sensitivities to Zn²⁺ inhibition. However, GlyR α2β heteromers were consistently 2-fold more sensitive to inhibition compared to the GlyR α2 homomer. Comparative studies to elucidate the specific residue in the β subunit responsible for this differential sensitivity revealed instead threonine 133 in the α1 subunit as a new vital component for Zn²⁺-mediated inhibition. Further studies on heteromeric receptors demonstrated that a mutated β subunit could indeed affect Zn²⁺-mediated inhibition but only from one side of the intersubunit Zn²⁺-binding site, equivalent to the GlyR α1 H107 face. This strongly suggests that the α subunit is responsible for Zn²⁺-mediated inhibition and that this is effectively transduced, asymmetrically, from the side of the Zn²⁺-binding site where H109 and T133 are located.     

Crucial function of the pre-T-cell receptor (TCR) in TCRP selection, TCRβ allelic exclusion and αβ versus γδ lineage commitment

Summary: The analysis of T-cell receptor (TCR) βselection, TCRβ allelic exclusion and TCRβ rearrangement in γδ T cells from normal and pre-TCR-deficient mice has shown that the pre-TCR has a crucial role in T-lyinpbocyte development:

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  The pre-TCR is by far the most effective receptor that generates large numbers of CD4⁺8⁺ T cells with productive TCRβ rearrangements.
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  In the absence of the pre-TCR, TCRβ rearrangement proceeds in developing cells irrespective of whether they already contain a productive TCRβ gene.
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  The pre-TCR directs developing T cells to the αβ lineage because y5 T cells from pTα^-/- mice proceed much further in TCRβ rearrangement than γδ T cells from wild-type mice. It is argued that the pre-TCR commits developing T cells to the αβ lineage by an instructive mechanism, which has largely replaced an evolutionarily more ancient mechanism that involves stochastic αβ lineage commitment.

     

Human NK Cells Expressing α4β1/β7 Adhere to VCAM-1 Without Preactivation

The authors demonstrate that resting CD56⁺/CD3⁻ NK cell adhesion to the endothelial VCAM-1 is over three-fold higher than CD56⁻/CD3 ⁺ T-cell adhesion. T-cell, but not NK-cell adhesion, to VCAM-1 is enhanced significantly by stimulation. The expression of VCAM-I receptor subunits α4 and β1 on both effector cells remains unchanged upon stimulation. A subpopulation of NK cells, as well as of T cells, was found to express β7, whose expression was not altered upon stimulation. The authors conclude that the adhesive properties of the same receptor structures on these distinct cell populations are regulated in a different manner, according to the specific functions of the effector cells of the immune system.     

Absence of humoral mediated 5'AMP-activated protein kinase activation in human skeletal muscle and adipose tissue during exercise

5'AMP-activated protein kinase (AMPK) exists as a heterotrimer comprising a catalytic α subunit and regulatory β and γ subunits. The AMPK system is activated under conditions of cellular stress, indicated by an increase in the AMP/ATP ratio, as observed, e.g. in muscles during contractile activity. AMPK was originally thought to be activated only by local intracellular mechanisms. However, recently it has become apparent that AMPK in mammals is also regulated by humoral substances, e.g. catecholamines. We studied whether humoral factors released during exercise regulate AMPK activity in contracting and resting muscles as well as in abdominal subcutaneous adipose tissue in humans. In resting leg muscle and adipose tissue the AMPK activity was not up-regulated by humoral factors during one-legged knee extensor exercise even when arm cranking exercise, inducing a ∼20-fold increase in plasma catecholamine level, was added simultaneously. In exercising leg muscle the AMPK activity was increased by one-legged knee extensor exercise eliciting a whole body respiratory load of only 30%     but was not further increased by adding arm cranking exercise. In conclusion, during exercise with combined leg kicking and arm cranking, the AMPK activity in human skeletal muscle is restricted to contracting muscle without influence of marked increased catecholamine levels. Also, with this type of exercise the catecholamines or other humoral factors do not seem to be physiological regulators of AMPK in the subcutaneous adipose tissue.     

A Highly Conserved Phenylalanine in the α, β-T Cell Receptor (TCR) Constant Region Determines the Integrity of TCR/CD3 Complexes

In the present study, we have investigated the importance of a phenylalanine (phe¹⁹⁵) in the Tcr-Cα region on Tcr-α, β/CD3 membrane expression. An exchange of phe¹⁹⁵ with a tyrosine residue does not affect Tcr/CD3 membrane expression; however, exchange with aspartic acid, histidine or valine prohibit completely Tcr/CD3 membrane expression. This seems to be due to a lack of interaction between mutated Tcr-α, β/CD3-γɛ, δɛ complexes and ζ₂ homodimers. The Tcr-Cα region around phe¹⁹⁵ seems together with the same region in the Tcr-Cβ region to constitute an interaction site for ζ₂ homodimers. The presence of phe¹⁹⁵ on both Tcr-Cα and Tcr-Cβ causes high avidity interaction with ζ₂ homodimers, whereas his¹⁹⁵ in both Tcr-Cγ and Tcr-Cδ results in an apparently lower avidity interaction with ζ₂ homodimers. It is suggested that the phe¹⁹⁵ region (on β-strand F) and eventually adjacent aromatic amino acid residues on β-strand B region may play an important role in Tcr-α, β/CD3 membrane expression, in Tcr-α, β/CD3 competition with Tcr-γ, δ/CD3 complexes for ζ₂ homodimers and in the control of formation of 'mixed' Tcr heterodimers.     

Intracellular Na+ inhibits voltage-dependent N-type Ca2+ channels by a G protein βγ subunit-dependent mechanism

N-type  voltage-dependent  Ca²⁺ channels (N-VDCCs) play important roles in neurotransmitter release and certain postsynaptic phenomena. These channels are modulated by a number of intracellular factors, notably by Gβγ subunits of G proteins, which inhibit N-VDCCs in a voltage-dependent (VD) manner. Here we show that an increase in intracellular Na⁺ concentration inhibits N-VDCCs  in hippocampal pyramidal neurones and in Xenopus oocytes. In acutely dissociated hippocampal neurones, Ba²⁺ current via N-VDCCs was inhibited by Na⁺ influx caused by the activation of NMDA receptor channels. In Xenopus oocytes expressing N-VDCCs, Ba²⁺ currents were inhibited by Na⁺ influx and enhanced by depletion of Na⁺, after incubation in a Na⁺-free extracellular solution. The Na⁺-induced inhibition was accompanied by the development of  VD facilitation, a hallmark of a Gβγ-dependent process. Na⁺-induced regulation of N-VDCCs is Gβγ dependent, as suggested by the blocking of Na⁺ effects by Gβγ scavengers and by excess Gβγ, and may be mediated by the Na⁺-induced dissociation of Gαβγ heterotrimers. N-VDCCs may be novel effectors of Na⁺ion, regulated by the Na⁺ concentration via Gβγ.     

Fatty acids stimulate AMP-activated protein kinase and enhance fatty acid oxidation in L6 myotubes

We investigated the role of fatty acid availability on AMPK signalling and fatty acid oxidation in skeletal muscle. Incubating L6 skeletal muscle myotubes with palmitate (a saturated fatty acid) or linoleate (a polyunsaturated fatty acid) increased AMPK activity by 56 and 38%, respectively, compared with untreated cells. Consistent with these changes, AMPK Thr¹⁷² and acetyl-CoA carboxylase β Ser²¹⁸ phosphorylation were increased in fatty acid treated cells. Pre-incubating cells with palmitate or linoleate increased subsequent fatty acid oxidation by 86 and 92%, respectively. The enhanced AMPK signalling occurred in the absence of detectable changes in free AMP and glycogen content. The activity of the upstream kinase LKB1 was decreased by fatty acid treatment indicating that AMPK activation was not a consequence of LKB1 activation. Instead, fatty acids enhanced LKB1 phosphorylation of AMPK. Fatty acids did not alter LKB1 activity when either synthetic peptide or AMPK α(1–312) catalytic fragment was used as substrate indicating that the βγ subunits were required for the fatty acid activation. Infection of cells with a dominant-negative AMPK adenovirus reduced basal fatty acid oxidation and inhibited the stimulatory effects of fatty acid pretreatment on fatty acid oxidation. These results indicate that increasing fatty acid availability increases AMPK activity independent of changes in the cellular energy charge and support the view that fatty acids may modulate AMPK allosterically, making it a better substrate for LKB1.     

Vδ1+ Gamma/Delta T Lymphocytes Infiltrating Human Lung Cancer Express the CD8α/α Homodimer

Murine γ/δ T lymphocytes localize to different epithelial tissues and are phenotypically distinct from peripheral γ/δ T cell-populations in that they show limited TCR diversity, express the CD8 α/α homodimer and lack the CD8β chain. In humans, a compartmentalization of γ/δ cells sharing similar phenotypic features has been documented to date only in the case of intestinal epithelium. In the present study we show that about half of Vδ1⁺ (as well as Vδ1⁻Vδ2⁻) γ/δ lymphocytes, which can be selectively expanded from human lung cancers, coexpress the CD8α/α homodimer. The accumulation of intraepithelial CD8⁺γ/δ⁺ lymphocytes might then be a more general phenomenon, possibly as a result of common mechanisms operating at those sites.     

Effect of endurance exercise training on Ca2+–calmodulin-dependent protein kinase II expression and signalling in skeletal muscle of humans

Here the hypothesis that skeletal muscle Ca²⁺–calmodulin-dependent kinase II (CaMKII) expression and signalling would be modified by endurance training was tested. Eight healthy, young men completed 3 weeks of one-legged endurance exercise training with muscle samples taken from both legs before training and 15 h after the last exercise bout. Along with an ∼40% increase in mitochondrial F₁-ATP synthase expression, there was an ∼1-fold increase in maximal CaMKII activity and CaMKII kinase isoform expression after training in the active leg only. Autonomous CaMKII activity and CaMKII autophosphorylation were increased to a similar extent. However, there was no change in α-CaMKII anchoring protein expression with training. Nor was there any change in expression or Thr¹⁷ phosphorylation of the CaMKII substrate phospholamban with training. However, another CaMKII substrate, serum response factor (SRF), had an ∼60% higher phosphorylation at Ser¹⁰³ after training, with no change in SRF expression. There were positive correlations between the increases in CaMKII expression and SRF phosphorylation as well as F₁ATPase expression with training. After training, there was an increase in cyclic-AMP response element binding protein phosphorylation at Ser¹³³, but not expression, in muscle of both legs. Taken together, skeletal muscle CaMKII kinase isoform expression and SRF phosphorylation is higher with endurance-type exercise training, adaptations that are restricted to active muscle. This may contribute to greater Ca²⁺ mediated regulation during exercise and the altered muscle phenotype with training.     

Tyrosine kinases enhance the function of glycine receptors in rat hippocampal neurons and human α1β glycine receptors

Glycine receptors (GlyRs) are transmitter-gated channels that mediate fast inhibitory neurotransmission in the spinal cord and brain. The GlyR β subunit contains a putative tyrosine phosphorylation site whose functional role has not been determined. To examine if protein tyrosine kinases (PTKs) regulate the function of GlyRs, we analysed whole-cell currents activated by applications of glycine to CA1 hippocampal neurons and spinal neurons. The role of a putative site for tyrosine phosphorylation at position 413 of the β subunit was examined using site-directed mutagenesis and expression of recombinant (α₁β^Y413F ) receptors in human embryonic kidney (HEK 293) cells. Lavendustin A, an inhibitor of PTKs, depressed glycine-evoked currents ( I _Gly) in CA1 neurons and spinal neurons by 31 % and 40 %, respectively. In contrast, the intracellular application of the exogenous tyrosine kinase, cSrc, enhanced I _Gly in CA1 neurons by 56 %. cSrc also accelerated GlyR desensitization and increased the potency of glycine 2-fold (control EC₅₀= 143 μ m ; cSrc EC₅₀= 74 μ m ). Exogenous cSrc, applied intracellularly, upregulated heteromeric α₁β receptors but not homomeric α₁ receptors. Substitution mutation of the tyrosine to phenylalanine at position β-413 prevented this enhancement. Furthermore, a selective inhibitor of the Src family kinases, PP2, down-regulated wild-type α₁β but not α₁β^Y413F receptors. Together, these findings indicate that GlyR function is upregulated by PTKs and this modulation is dependent on the tyrosine-413 residue of the β subunit.     

CD3−4−8− Thymocyte Precursors with Interleukin-2 Receptors Differentiate Phenotypically in Coculture with Thymic Stromal Cells

CD3⁻4⁻8⁻ interleukin-2 receptor positive (IL-2R⁺) thymocyte precursors from adult mice were cocultured with thymic stromal cells from syngeneic adult mice. The IL-2R⁺CD3⁻4⁻8⁻ thymocytes were obtained by positive panning of IL-2R⁺ cells followed by either sorting or negative panning of triple negative cells, and they were cocultured with primary or secondary cultures of heterogeneous thymic stromal cells. Phenotypic maturation of these precursor cells was extremely rapid. Within 2½ days significant numbers of CD4⁺8⁺ and CD3⁺4⁺8⁻ cell populations developed, the latter expressing the αβ T-cell receptor (αβ-TCR). Thus heterogeneous stromal cell cultures support the development of IL-2R⁺ precursors and with these methods it will now be possible to isolate the particular stromal cells involved at each stromal-dependent step.     

Ca2+–calmodulin-dependent protein kinase expression and signalling in skeletal muscle during exercise

Ca²⁺ signalling is proposed to play an important role in skeletal muscle function during exercise. Here, we examined the expression of multifunctional Ca²⁺–calmodulin-dependent protein kinases (CaMK) in human skeletal muscle and show that CaMKII and CaMKK, but not CaMKI or CaMKIV, are expressed. Furthermore, the effect of exercise duration and intensity on skeletal muscle CaMKII activity and phosphorylation of downstream targets was examined. Eight healthy men exercised at ∼67% of peak pulmonary O₂ uptake     with muscle samples taken at rest and after 1, 10, 30, 60 and 90 min of exercise. Ten other men exercised for three consecutive 10 min bouts at 35%, 60% and 85%     with muscle samples taken at rest, at the end of each interval and 30 min post-exercise. There was a rapid and transient increase in autonomous CaMKII activity and CaMKII phosphorylation at Thr²⁸⁷ in skeletal muscle during exercise. Furthermore, the phosphorylation of phospholamban (PLN) at Thr¹⁷, which was identified as a CaMKII substrate in skeletal muscle, was rapidly (< 1 min) increased by exercise, and remained phosphorylated 5-fold above basal level during 90 min of exercise. The phosphorylation of serum response factor at Ser¹⁰³, a putative CaMKII substrate, was higher after 30 min of exercise. PLN phosphorylation at Thr¹⁷ was higher with increasing exercise intensities. These data indicate that CaMKII is the major multifunctional CaMK in skeletal muscle and its activation occurs rapidly and is sustained during continuous exercise, with the activation being greater during intense exercise.