Mechanical stimuli and nutrients regulate rapamycin-sensitive signaling through distinct mechanisms in skeletal muscle

The mammalian target of rapamycin (mTOR) has been identified as a growth factor and nutrient-sensitive molecule that controls the translational machinery and cell growth. Rapamycin-sensitive (RS) signaling events have also been shown to be necessary for mechanical load-induced growth of skeletal muscle, but the mechanisms involved in the mechanical activation of RS signaling are not known. The finding that mechanical stimuli induce nutrient uptake in skeletal muscle raises the possibility that mechanically induced RS signaling is mediated via a nutrient-dependent mechanism. To investigate this hypothesis, skeletal muscles (ex vivo) were stimulated with nutrients or intermittent mechanical stretch and the phosphorylation of p70S6k [P-p70(389)], PKB [P-PKB], mTOR [P-mTOR(2481)], and p38 [P-p38] was assessed. In comparison to vehicle-treated controls, both nutrient and mechanical stimuli induced P-p70(389), neither stimulus altered P-PKB or P-mTOR(2481), and only mechanical stimuli induced P-p38. The nutrient and mechanically induced increase in P-p70(389) was blocked by rapamycin, but only nutrient-induced signaling to P-p70(389) was blocked by wortmannin. Furthermore, the mechanically induced increase in P-p70(389) was not impaired by the removal of exogenous nutrients. Taken together, these results indicate that exogenous nutrients are not required for mechanically induced RS signaling and that nutrient and mechanical stimuli activate RS signaling through distinct upstream mechanisms.     

Static stretch increases c-Jun NH2-terminal kinase activity and p38 phosphorylation in rat skeletal muscle

Physicalexercise and contraction increase c-Jun NH₂-terminal kinase(JNK) activity in rat and human skeletal muscle, and eccentriccontractions activate JNK to a greater extent than concentric contractions in human skeletal muscle. Because eccentric contractions include a lengthening or stretch component, we compared the effects ofisometric contraction and static stretch on JNK and p38, the stress-activated protein kinases. Soleus and extensor digitorum longus(EDL) muscles dissected from 50- to 90-g male Sprague-Dawley rats weresubjected to 10 min of electrical stimulation that produced contractions and/or to 10 min of stretch (0.24 N tension, 20-25% increase in length) in vitro. In the soleus muscle, contraction resulted in a small, but significant, increase in JNK activity (1.8-fold above basal) and p38 phosphorylation (4-fold). Static stretchhad a much more profound effect on the stress-activated proteinkinases, increasing JNK activity 19-fold and p38 phosphorylation 21-fold. Increases in JNK activation and p38 phosphorylation in response to static stretch were fiber-type dependent, with greater increases occurring in the soleus than in the EDL. Immunohistochemistry performed with a phosphospecific antibody revealed that activation ofJNK occurred within the muscle fibers. These studies suggest that thestretch component of a muscle contraction may be a major contributor tothe increases in JNK activity and p38 phosphorylation observed afterexercise in vivo.

     

Altered activity of signaling pathways in diaphragm and tibialis anterior muscle of dystrophic mice

Duchenne muscular dystrophy is a musculoskeletal disease caused by mutations in the dystrophin gene. The purpose of this study was to use the mouse model of muscular dystrophy (mdx) to determine if the progression of the dystrophic phenotype in the diaphragm (costal) versus limb skeletal muscle (tibialis anterior) is associated with specific changes in extracellular regulated kinase (ERK1/2), p70 S6 kinase (p70(S6k)), or p38 signaling pathways. The studies detected that consistent with an earlier dystrophic phenotype, phosphorylation of p70(S6k) is elevated by 40% in the diaphragm with no change in limb muscle. In addition, phosphorylation of p38 kinase was decreased by 33% in the mdx diaphragm muscle. Levels of ERK1/2 as well as phosphorylation states were elevated in the diaphragm and limb muscle of mdx mice compared with age-matched control muscles. These results indicate that distinct signaling pathways are differentially activated in skeletal muscle of mdx mice. The specificity of these responses, particularly in the diaphragm, provides insight for potential targets for blunting the progression of the muscular dystrophy phenotype.     

Autocrine Phosphorylation of p70S6k in Response to Acute Stretch in Myotubes

Phosphorylation of 70-KDa S6 kinase (p70^S6k) is correlated with in vivo skeletal muscle hypertrophy. Experiments tested whether mechanical stretch is sufficient to increase p70^S6k phosphorylation in skeletal myotubes. Immediately following stretch, there was a small increase in p70^S6k phosphorylation (63.2 ± 8.5%) with maximal phosphorylation at 3 h (129.5 ± 22.2%) and it remained elevated through 24 h (46.0 ± 17.2%). To test whether an autocrine mechanism is involved, unstretched myotubes were incubated with medium from the stretch group for 10 min. Conditioned medium resulted in the phosphorylation of p70^S6k in unstretched myotubes (92.8 ± 28.9%) to levels comparable to the 3-h stretch group. These data indicate that p70^S6k is phosphorylated in stretched myotubes via a mechanism that most likely involves an autocrine signaling pathway.     

Uncoupling protein-2 polymorphisms in type 2 diabetes,obesity, and insulin secretion

The aim of the study was to investigate the effect of resistance exercise alone or in combination with oral intake of branched-chain amino acids (BCAA) on phosphorylation of the 70-kDa S6 protein kinase (p70(S6k)) and mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK1/2), and p38 MAPK in skeletal muscle. Seven male subjects performed one session of quadriceps muscle resistance training (4 x 10 repetitions at 80% of one repetition maximum) on two occasions. In a randomized order, double-blind, crossover test, subjects ingested a solution of BCAA or placebo during and after exercise. Ingestion of BCAA increased plasma concentrations of isoleucine, leucine, and valine during exercise and throughout recovery after exercise (2 h postexercise), whereas no change was noted after the placebo trial. Resistance exercise led to a robust increase in p70(S6k) phosphorylation at Ser(424) and/or Thr(421), which persisted 1 and 2 h after exercise. BCAA ingestion further enhanced p70(S6k) phosphorylation 3.5-fold during recovery. p70(S6k) phosphorylation at Thr(389) was unaltered directly after resistance exercise. However, during recovery, Thr(389) phosphorylation was profoundly increased, but only during the BCAA trial. Furthermore, phosphorylation of the ribosomal protein S6 was also increased in the recovery period only during the BCAA trial. Exercise led to a marked increase in ERK1/2 and p38 MAPK phosphorylation, which was completely suppressed upon recovery and unaltered by BCAA. In conclusion, BCAA, ingested during and after resistance exercise, mediate signal transduction through p70(S6k) in skeletal muscle.     

Mechanical stretch activates signaling events for protein translation initiation and elongation in C2C12 myoblasts

It has been proposed that mechanically induced tension is the critical factor in the induction of muscle hypertrophy. However, the molecular mechanisms involved in this process are still under investigation. In the present study, the effect of mechanical stretch on intracellular signaling for protein translation initiation and elongation was studied in C2C12 myoblasts. Cells were grown on a silicone elastomer chamber and subjected to 30-min of 5 or 15% constant static or cyclic (60 cycles/min) uniaxial stretch. Western blot analyses revealed that p70 S6 kinase (p70S6K) and eukaryotic elongation factor 2 (eEF2), which are the markers for translation initiation and peptide chain elongation, respectively, were activated by both static and cyclic stretch. The magnitude of activation was greater in response to the 15% cyclic stretch. Cyclic stretch also increased the phosphorylation of MAP kinases (p38 MAPK, ERK1/2 and JNK). However, the pharmacological inhibition of MAP kinases did not block the stretch-induced activation of p70S6K and eEF2. An inhibitor of the mammalian target of rapamycin (mTOR) blocked the stretch-induced phosphorylation of p70S6K but did not affect the eEF2 activation. A broad-range tyrosine kinase inhibitor, genistein, blocked the stretch-induced activation of p70S6K and eEF2, whereas Src tyrosine kinase and Janus kinase (JAK) inhibitors did not. These results suggest that the stretch-induced activation of protein translation initiation and elongation in mouse myoblast cell lines is mediated by tyrosine kinase(s), except for Src kinase or JAK.     

Overexpression or ablation of JNK in skeletal muscle has no effect on glycogen synthase activity

c-Jun NH₂-terminal kinase (JNK) is highly expressed in skeletal muscle and is robustly activated in response to muscle contraction. Little is known about the biological functions of JNK signaling in terminally differentiated muscle cells, although this protein has been proposed to regulate insulin-stimulated glycogen synthase activity in mouse skeletal muscle. To determine whether JNK signaling regulates contraction-stimulated glycogen synthase activation, we applied an electroporation technique to induce JNK overexpression (O/E) in mouse skeletal muscle. Ten days after electroporation, in situ muscle contraction increased JNK activity 2.6-fold in control muscles and 15-fold in the JNK O/E muscles. Despite the enormous activation of JNK activity in JNK O/E muscles, contraction resulted in similar increases in glycogen synthase activity in control and JNK O/E muscles. Consistent with these findings, basal and contraction-induced glycogen synthase activity was normal in muscles of both JNK1- and JNK2-deficient mice. JNK overexpression in muscle resulted in significant alterations in the basal phosphorylation state of several signaling proteins, such as extracellular signal-regulated kinase 1/2, p90 S6 kinase, glycogen synthase kinase 3, protein kinase B/Akt, and p70 S6 kinase, in the absence of changes in the expression of these proteins. These data suggest that JNK signaling regulates the phosphorylation state of several kinases in skeletal muscle. JNK activation is unlikely to be the major mechanism by which contractile activity increases glycogen synthase activity in skeletal muscle. electroporation; gene delivery; muscle contraction; exercise     

Improved insulin sensitivity with calorie restriction does not require reduced JNK1/2, p38, or ERK1/2 phosphorylation in skeletal muscle of 9-month-old rats

Calorie restriction [CR; ～40% below ad libitum (AL) intake] improves the health of many species, including rats, by mechanisms that may be partly related to enhanced insulin sensitivity for glucose disposal by skeletal muscle. Excessive activation of several mitogen-activated protein kinases (MAPKs), including JNK1/2, p38, and ERK1/2 has been linked to insulin resistance. Although insulin can activate ERK1/2, this effect is not required for insulin-mediated glucose uptake. We hypothesized that skeletal muscle from male 9-mo-old Fischer 344/Brown Norway rats CR (35-40% beginning at 3 mo old) versus AL rats would have 1) attenuated activation of JNK1/2, p38, and ERK1/2 under basal conditions; and 2) no difference for insulin-induced ERK1/2 activation. In contrast to our hypothesis, there were significant CR-related increases in the phosphorylation of p38 (epitrochlearis, soleus, and gastrocnemius), JNK1 (epitrochlearis and soleus), and JNK2 (gastrocnemius). Consistent with our hypothesis, CR did not alter insulin-mediated ERK1/2 activation. The greater JNK1/2 and p38 phosphorylation with CR was not attributable to diet effects on muscle oxidative stress (assessed by protein carbonyls and 4-hydroxynonenal protein conjugates). In muscles from the same rats used for the present study, we previously reported a CR-related increase in insulin-mediated glucose uptake by the epitrochlearis and the soleus (Sharma N, Arias EB, Bhat AD, Sequea DA, Ho S, Croff KK, Sajan MP, Farese RV, Cartee GD. Am J Physiol Endocrinol Metab 300: E966-E978, 2011). The present results indicate that the improved insulin sensitivity with CR is not attributable to attenuated MAPK phosphorylation in skeletal muscle.     

Mechanism of zinc-induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3beta in SH-SY5Y neuroblastoma cells

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)-3beta, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK-3beta phosphorylation is unknown. In the current study, 100 microM zinc sulfate was used, and influences of various components of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on p70S6K and GSK-3beta phosphorylation have been investigated in serum-deprived SH-SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p-PKB, p-GSK-3beta, p-ERK1/2, p-JNK and p-p38, especially in long-term treatment (4-8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK-3beta is regulated by rapamycin-dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK-3beta affected levels of tau unphosphorylated at the Tau-1 site and phosphorylated at the PHF-1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 microM zinc might activate PKB, GSK-3beta, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH-SY5Y cells.     

Contraction-mediated mTOR, p70S6k, and ERK1/2 phosphorylation in aged skeletal muscle.

With age, skeletal muscle experiences substantial atrophy and weakness. Although resistance training can increase muscle size and strength, the myogenic response to exercise and the capacity for muscle hypertrophy in older humans and animals is limited. In the present study, we assessed the ability of muscle contractile activity to activate cellular pathways involved in muscle cell growth and myogenesis in adult (Y; 6 mo old) and aged (O; 30 mo old) Fischer 344 x Brown Norway rats. A single bout of rat hindlimb muscle contractile activity was elicited by high-frequency electrical stimulation (HFES) of the sciatic nerve. Plantaris (Pla) and tibialis anterior (TA) muscles were assayed for mammalian target of rapamycin (mTOR), 70-kDa ribosomal protein S6 kinase (p70(S6K)), and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and total protein either at baseline, immediately after, or 6 h after HFES. mTOR phosphorylation was elevated in Pla (1.3 +/- 0.3-fold, P < 0.05) immediately after HFES and to a lesser extent 6 h after HFES (0.6 +/- 0.1-fold, P < 0.05) in O rats. Post-HFES, p70(S6K) phosphorylation increased 1.2 +/- 0.3-fold in TA (P < 0.05) and remained elevated 6 h later (0.6 +/- 0.2-fold, P < 0.05) in O rats. ERK phosphorylation was lower in O rats immediately after exercise in both TA (11.1 +/- 2.9 vs. 2.1 +/- 0.5-fold, P < 0.05) and Pla (6.5 +/- 1.5 vs. 1.8 +/- 0.5-fold, P < 0.05) and returned to baseline by 6 h in both Y and O rats. Phosphorylation of mTOR, p70(S6K), and ERK1/2 are increased in skeletal muscle after a single bout of in situ muscle contractile activity in aged animals, and the response is less than that observed in adult animals. These observations suggest that the anabolic response to a single bout of contraction is attenuated with aging and may help explain the reduced capacity for hypertrophy in aged animals.     

Free fatty acid-induced muscle insulin resistance and glucose uptake dysfunction: Evidence for PKC activation and oxidative stress-activated signaling pathways

In the present study, we examined the effects of free fatty acids (FFAs) on insulin sensitivity and signaling cascades in the C2C12 skeletal muscle cell culture system. Our data clearly manifested that the inhibitory effects of PKC on insulin signaling may at least in part be explained by the serine/threonine phosphorylation of IRS-1. Both oleate and palmitate treatment were able to increase the Serine³⁰⁷ phosphorylation of IRS-1. IRS-1 Serine³⁰⁷ phosphorylation is inducible which causes the inhibition of IRS-1 tyrosine phosphorylation by either IκB-kinase (IKK) or c-jun N-terminal kinase (JNK) as seen in our proteomic kinases screen. Furthermore, our proteomic data have also manifested that the two FFAs activate the IKKα/β, the stress kinases S6 kinase p70 (p70SK), stress-activated protein kinase (SAPK), JNK, as well as p38 MAP kinase (p38MAPK). On the other hand, the antioxidant, Taurine at 10 mM concentrations was capable of reversing the oleate-induced insulin resistance in myocytes as manifested from the glucose uptake data. Our current data point out the importance of FFA-induced insulin resistance via multiple signaling mechanisms.     

mTOR is the rapamycin-sensitive kinase that confers mechanically-induced phosphorylation of the hydrophobic motif site Thr(389) in p70(S6k)

Mechanical stretch induces phosphorylation of the hydrophobic motif site Thr(389) in p70(S6k) through a rapamycin-sensitive (RS) pathway that involves a unique PI3K-independent mechanism. Rapamycin is considered to be a highly specific inhibitor of the protein kinase mTOR; however, mTOR is also considered to be a PI3K-dependent signaling molecule. Thus, questions remain as to whether mTOR is the RS element that confers mechanically-induced signaling to p70(S6k)(389). In this study, rapamycin-resistant mutants of mTOR along with mechanical stretch were used to address this question. The results indicate that mTOR is the RS element and reveal that mTOR signaling can be activated through a PI3K-independent mechanism.     

Cyclic mechanical stretch stimulates the proliferation of C2C12 myoblasts and inhibits their differentiation via prolonged activation of p38 MAPK

Mitogen-activated protein kinases (MAPKs) play an indispensable role in activation of the myogenic program, which is responsive to mechanical stimulation. Although there is accumulating evidence of mechanical force-mediated cellular responses, the role of MAPK in regulating the myogenic process in myoblasts exposed to cyclic stretch is unclear. Cyclic stretch induced the proliferation of C2C12 myoblasts and inhibited their differentiation into myotubes. In particular, it induced persistent phosphorylation of p38 kinase, and decreased the level of phosphorylation of extracellular-signal regulated kinase (ERK). Partial inhibition of p38 phosphorylation increased cellular levels of MyoD and p-ERK in stretched C2C12 cells, along with increased myotube formation. Treatment with 10 microM PD98059 prevented myogenin expression in response to a low dose of SB203580 (3 microM) in the stretched cells, suggesting that adequate ERK activation is also needed to allow the cells to differentiate into myotubes. These results suggest that cyclic stretch inhibits the myogenic differentiation of C2C12 cells by activating p38-mediated signaling and inhibiting ERK phosphorylation. We conclude that p38 kinase, not ERK, is the upstream signal transducer regulating cellular responses to mechanical stretch in skeletal muscle cells.     

Leucine and insulin activate p70 S6 kinase through different pathways in human skeletal muscle

Amino acids and insulin have anabolic effects in skeletal muscle, but the mechanisms are poorly understood. To test the hypothesis that leucine and insulin stimulate translation initiation in human skeletal muscle by phosphorylating 70-kDa ribosomal protein S6 kinase (p70(S6k)), we infused healthy adults with leucine alone (n = 6), insulin alone (n = 6), or both leucine and insulin (n = 6) for 2 h. p70(S6k) and protein kinase B (PKB) serine(473) phosphorylation were measured in vastus lateralis muscles. Plasma leucine increased from approximately 116 to 343 micromol/l during the leucine-alone and leucine + insulin infusions. Plasma insulin increased to approximately 400 pmol/l during the insulin-alone and leucine + insulin infusions and was unchanged during the leucine-alone infusion. Phosphorylation of p70(S6k) increased 4-fold in response to leucine alone, 8-fold in response to insulin alone, and 18-fold after the leucine + insulin infusion. Insulin-alone and leucine + insulin infusions increased PKB phosphorylation, but leucine alone had no effect. These results show that physiological concentrations of leucine and insulin activate a key mediator of protein synthesis in human skeletal muscle. They suggest that leucine stimulates protein synthesis through a nutrient signaling mechanism independent of insulin, raising the possibility that administration of branched-chain amino acids may improve protein synthesis in insulin-resistant states.     

Insight into skeletal muscle mechanotransduction: MAPK activation is quantitatively related to tension.

The mechanism by which mechanical forces acting through skeletal muscle cells generate intracellular signaling, known as mechanotransduction, and the details of how gene expression and cell size are regulated by this signaling are poorly understood. Mitogen-activated protein kinases (MAPKs) are known to be involved in mechanically induced signaling in various cell types, including skeletal muscle where MAPK activation has been reported in response to contraction and passive stretch. Therefore, the investigation of MAPK activation in response to mechanical stress in skeletal muscle may yield important information about the mechanotransduction process. With the use of a rat plantaris in situ preparation, a wide range of peak tensions was generated through passive stretch and concentric, isometric, and eccentric contractile protocols, and the resulting phosphorylation of c-Jun NH(2)-terminal kinase (JNK), extracellular regulated kinase (ERK), and p38 MAPKs was assessed. Isoforms of JNK and ERK MAPKs were found to be phosphorylated in a tension-dependent manner, such that eccentric > isometric > concentric > passive stretch. Peak tension was found to be a better predictor of MAPK phosphorylation than time-tension integral or rate of tension development. Differences in maximal response amplitude and sensitivity between JNK and ERK MAPKs suggest different roles for these two kinase families in mechanically induced signaling. A strong linear relationship between p54 JNK phosphorylation and peak tension over a 15-fold range in tension (r(2) = 0.89, n = 32) was observed, supporting the fact that contraction-type differences can be explained in terms of tension and demonstrating that MAPK activation is a quantitative reflection of the magnitude of mechanical stress applied to muscle. Thus the measurement of MAPK activation, as an assay of skeletal muscle mechanotransduction, may help elucidate mechanically induced hypertrophy.     

Phosphorylation and degradation of S6K1 (p70S6K1) in response to persistent JNK1 Activation

S6K (ribosomal S6 kinase p70, p70S6K) activation requires phosphorylation at two stages. The first phosphorylation is independent of insulin stimulation and mediated by an unknown kinase. The second phosphorylation is mediated by mTOR in insulin dependent manner. In this study, we identified JNK1 (c-Jun N-terminal kinase 1) as a kinase in the first phosphorylation. S6K protein was phosphorylated by JNK1 at S411 and S424 in the carboxyl terminal autoinhibitory domain. The phosphorylation was observed in kinase assay with purified S6K as a substrate, and in cells after JNK1 activation by TNF-α or MEKK1 expression. The phosphorylation was detected in JNK2 null cells, but not in JNK1 null cells after TNF-α treatment. When JNK1 activation was inhibited by MKK7 knockdown, the phosphorylation was blocked in cells. The phosphorylation led to S6K protein degradation in NF-κB deficient cells. The degradation was blocked by inhibition of proteasome activity with MG132. In wide type cells, the phosphorylation did not promote S6K degradation when IKK2 (IKKβ, IκB kinase beta) was activated. Instead, the phosphorylation allowed S6K activation by mTOR, which stabilizes S6K protein. In IKK2 null cells or cells treated by IKK2 inhibitor, the phosphorylation led to S6K degradation. These data suggest that S6K is phosphorylated by JNK1 and the phosphorylation makes S6K protein unstable in the absence of IKK2 activation. This study provides a mechanism for regulation of S6K protein stability.     

An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase.

This study characterizes the insulin-activated serine/threonine protein kinases in H4 hepatoma cells active on a 37-residue synthetic peptide (called the SKAIPS peptide) corresponding to a putative autoinhibitory domain in the carboxyl-terminal tail of the p70 S6 kinase as well as on recombinant p70 S6 kinase. Three peaks of insulin-stimulated protein kinase active on both these substrates are identified as two (possibly three) isoforms of the 40-45-kDa erk/microtubule-associated protein (MAP)-2 kinase family and a 150-kDa form of cdc2. Although distinguishable in their substrate specificity, these protein kinases together with the p54 MAP-2 kinase share a major common specificity determinant reflected in the SKAIPS peptide: the requirement for a proline residue immediately carboxyl-terminal to the site of Ser/Thr phosphorylation. In addition, however, at least one peak of insulin-stimulated protein kinase active on recombinant p70, but not on the SKAIPS peptide, is present although not yet identified. MFP/cdc2 phosphorylates both rat liver p70 S6 kinase and recombinant p70 S6 kinase exclusively at a set of Ser/Thr residues within the putative autoinhibitory (SKAIPS peptide) domain. erk/MAP kinase does not phosphorylate rat liver p70 S6 kinase, but readily phosphorylates recombinant p70 S6 kinase at sites both within and in addition to those encompassed by the SKAIPS peptide sequences. Although the tryptic 32P-peptides bearing the cdc2 and erk/MAP kinase phosphorylation sites co-migrate with a subset of the sites phosphorylated in situ in insulin-stimulated cells, phosphorylation of the p70 S6 kinase by these proline-directed protein kinases in vitro does not reproducibly activate p70 S6 kinase activity. Thus, one or more erk/MAP kinases and cdc2 are likely to participate in the insulin-induced phosphorylation of the p70 S6 kinase. In addition to these kinases, however, phosphorylation of the p70 S6 kinase by other as yet unidentified protein kinases is necessary to recapitulate the multisite phosphorylation required for activation of the p70 S6 kinase.