Recent progress in characterization of protein kinase cascades for phosphorylation of ribosomal protein S6.

Ribosomal protein S6 is phosphorylated in response to mitogens by activation of one or more protein kinase cascades. Phosphorylation of S6 in vivo is catalyzed by (at least) two distinct mitogen-activated S6 kinase families distinguishable by size, the 70 kDa and 90 kDa S6 kinases. Both S6 kinases are activated by serine/threonine phosphorylation. Members of each family have been cloned. The 90 kDa S6 kinases are activated more rapidly than the 70 kDa S6 kinase, and may have other intracellular targets. The 70 kDa S6 kinase is relatively specific for 40 S ribosomal subunits. No kinase capable of activating the 70 kDa S6 kinase has been identified. Members of the 90 kDa S6 kinases are activated in vitro by 42 kDa and 44 kDa MAP kinases, which are in turn activated by mitogen-dependent activators. The pathways for mitogen-stimulated S6 phosphorylation are discussed.     

Molecular aspects of mechanical stress-induced cardiac hypertrophy

To elucidate the signal transduction pathway from external stimuli to nuclear gene expression in mechanical stress-induced cardiac hypertrophy, we examined the time course of activation of protein kinases such as Raf-1 kinase (Raf-1), mitogen-activated protein kinase kinase (MAPKK), MAP kinases (MAPKs) and 90-kDa ribosomal S6 kinase (p90^rsk) in neonatal rat cardiomyocytes. Mechanical stretch rapidly activated Raf-1 and its maximal activation was observed at 1–2 min after stretch. The activity of MAPKK was also increased by stretch, with a peak at 5 min after stretch. In addition, MAPKs and p90^rsk were maximally activated at 8 min and at 10–30 min after stretch, respectively. Next, the relationship between mechanical stress-induced hypertrophy and the cardiac renin-angiotensin system was investigated. When the stretch-conditioned culture medium was transferred to the culture dish of non-stretched cardiac myocytes, the medium activated MAPK activity slightly but significantly, and the activation was completely blocked by the type 1 angiotensin II receptor antagonist, CV-11974. However, activation of Raf-1 and MAPKs provoked by stretching cardiomyocytes was only partially suppressed by pretreatment with CV-11974. These results suggest that mechanical stress activates the protein kinase cascade of phosphorylation in cardiac myocytes in the order of Raf-1, MAPKK, MAPKs and p90^rsk, and that angiotensin II, which is secreted from stretched myocytes, activates a part of these protein kinases.Abbreviations MAPK mitogen-activated protein kinase - MAPKK MAP kinase kinase - Raf-1 - Raf- 1 kinase p90^rsk, 90 kDa ribosomal S6 kinase; AngII - angiotensin II - MAPKKK MAP kinase kinase kinase - rMAPK recombinant MAPKK fused to gluthathione S transferase - MMAKK recombinant MAPK fused to maltose binding protein - MBP myelin basic protein - ACE angiotensin-converting enzyme     

Vanadium salts stimulate mitogen-activated protein (MAP) kinases and ribosomal S6 kinases

Effect of several vanadium salts, sodium orthovanadate, vanadyl sulfate and sodium metavanadate on protein tyrosine phosphorylation and serine/threonine kinases in chinese hamster ovary (CHO) cells overexpressing a normal human insulin receptor was examined. All the compounds stimulated protein tyrosine phosphorylation of two major proteins with molecular masses of 42 kDa (p42) and 44 kDa (p44). The phosphorylation of p42 and p44 was associated with an activation of mitogen activated protein (MAP) kinase as well as increased protein tyrosine phosphorylation of p42^mapk and p44^mapk. Vanadinm salts also activated the 90 kDa ribosomal s6 kinase (p90^rsk) and 70 kDa ribosomal s6 kinase (p70^s6k). Among the three vanadium salts tested, vanadyl sulfate appeared to be slightly more potent than others in stimulating MAP kinases and p70^s6k activity. It is suggested that vanadium-induced activation of MAP kinases and ribosomal s6 kinases may be one of the mechanisms by which insulin like effects of this trace element are mediated.Abbreviations eIF-4 eukaryotic protein synthesis initiation factor-4 - GRB-2 growth factor receptor bound protein-2 - GSK-3 Glycogen Synthase Kinase-3 - IRS-1 insulin receptor substrate-1 - ISPK insulin stimulated protein kinase - MAPK mitogen activated protein kinase, also known as - ERK extracellular signal regulated kinase - MAPKK mitogen activated protein kinase kinase, also known as-MEK, MAPK or ERK kinase - PHAS-1 phosphorylated heat and acid stable protein regulated by insulin - PI3K phosphatidyl inositol 3-kinase - PP1-G protein phosphatase-glycogen bound form - PTK protein tyrosine kinase - PTPase protein tyrosine phosphatase - rsk ribosomal s6 kinases - shc src homology domain containing protein - SOS son of sevenless     

Stimulation of MAP kinase and S6 kinase by vanadium and selenium in rat adipocytes

To explore the mechanism underlying the insulin-mimetic actions of vanadium and selenium we examined their effects on the mitogen activated protein/myelin basic protein kinases (MAPK) and ribosomal S6 protein kinases, which are among the best characterized of the kinases that comprise the phosphorylation cascade in insulin signal transduction. We observed a transient activation of MAPK and S6 kinases by insulin in rat adipocytes, while both sodium selenate and vanadyl sulphate produced prolonged activation of the kinases. Vanadyl sulphate stimulated the activity of MAPK and S6 kinase by as much as 6 fold and 15 fold, respectively. Pretreatment of the cells with genistein did not affect the activation of MAPK by insulin, but partially blocked the effects of sodium selenate and vanadyl sulphate. Genistein did not change the activation of S6 kinase by insulin, but blocked the activation in vanadyl sulphate- and sodium selenate-treated-cells, suggesting that a genistein sensitive tyrosine kinase may be involved in the activation by these two compounds. Rapamycin, a specific inhibitor of the p70s6k isoform of S6 kinase, partially reduced the activation of S6 kinase activity by sodium selenate, indicating a role for this kinase in the overall activity of the S6 kinase in sodium selenate-treated cells. A similar trend was noted in vanadyl sulphate-treated cells. Thus, this study supports the involvement of MAPK and S6 kinases in the insulin-mimetic actions of vanadium and selenium.     

Interferon-gamma engages the p70 S6 kinase to regulate phosphorylation of the 40S S6 ribosomal protein

The signals generated by the IFNgamma receptor to initiate mRNA translation and generation of protein products that mediate IFNgamma responses are largely unknown. In the present study, we provide evidence for the existence of an IFNgamma-dependent signaling cascade activated downstream of the phosphatidylinositol (PI) 3'-kinase, involving the mammalian target of rapamycin (mTOR) and the p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated and activated during engagement of the IFNgamma receptor in sensitive cell lines. Such activation of p70 S6 kinase is blocked by pharmacological inhibitors of the PI 3' kinase and mTOR, and is abrogated in double-knockout mouse embryonic fibroblasts for the alpha and beta isoforms of the p85 regulatory subunit of the PI 3'-kinase. The IFNgamma-activated p70 S6 kinase subsequently phosphorylates the 40S S6 ribosomal protein on serines 235/236, to regulate IFNgamma-dependent mRNA translation. In addition to phosphorylation of 40S ribosomal protein, IFNgamma also induces phosphorylation of the 4E-BP1 repressor of mRNA translation on threonines 37/46, threonine 70, and serine 65, sites whose phosphorylation is required for the inactivation of 4E-BP1 and its dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. Thus, engagement of the PI 3'-kinase and mTOR by the IFNgamma receptor results in the generation of two distinct signals that play roles in the initiation of mRNA translation, suggesting an important role for this pathway in IFNgamma signaling.     

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.     

S6K1 is acetylated at lysine 516 in response to growth factor stimulation

The 70 kDa ribosomal protein S6 kinase 1 (S6K1) plays important roles in the regulation of protein synthesis, cell growth and metabolism. S6K1 is activated by the phosphorylation of multiple serine and threonine residues in response to stimulation by a variety of growth factors and cytokines. In addition to phosphorylation, we have recently shown that S6K1 is also targeted by lysine acetylation. Here, using tandem mass spectrometry we have mapped acetylation of S6K1 to lysine 516, a site close to the C-terminus of the kinase that is highly conserved amongst vertebrate S6K1 orthologues. Using acetyl-specific K516 antibodies, we show that acetylation of endogenous S6K1 at this site is potently induced upon growth factor stimulation. Although S6K1 acetylation and phosphorylation are both induced by growth factor stimulation, these events appear to be functionally independent. Indeed, experiments using inhibitors of S6K1 activation and exposure of cells to various stresses indicate that S6K1 acetylation can occur in the absence of phosphorylation and vice versa. We propose that K516 acetylation may serve to modulate important kinase-independent functions of S6K1 in response to growth factor signalling.     

Multiple implications of 3-phosphoinositide-dependent protein kinase 1 in human cancer

3-phosphoinositide-dependent protein kinase-1 (PDK1) is a central mediator of cellular signaling between phosphoinositide-3 kinase and various intracellular serine/threonine kinases, including protein kinase B, p70 ribosomal S6 kinase, serum and glucocorticoid-inducible kinase, and protein kinase C. PDK1 activates members of the AGC family of protein kinases by phosphorylating serine/threonine residues in the activation loop. Here, we review the regulatory mechanisms of PDK1 and its roles in cancer. PDK1 is activated by autophosphorylation in the activation loop and other serine residues, as well as by phosphorylation of Tyr-9 and Tyr-373/376. Src appears to recognize PDK1 following tyrosine phosphorylation. The role of heat shock protein 90 in regulating PDK1 stability and PDK1-Src complex formation are also discussed. Furthermore, we summarize the subcellular distribution of PDK1. Finally, an important role for PDK1 in cancer chemotherapy is proposed. In conclusion, a better understanding of its molecular regulatory mechanisms in various signaling pathways will help to explain how PDK1 acts as an oncogenic kinase in various cancers, and will contribute to the development of novel cancer chemotherapies.     

The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells

BACKGROUND: Protein kinase B (PKB), and the p70 and p90 ribosomal S6 kinases (p70 S6 kinase and p90 Rsk, respectively), are activated by phosphorylation of two residues, one in the 'T-loop' of the kinase domain and, the other, in the hydrophobic motif carboxy terminal to the kinase domain. The 3-phosphoinositide-dependent protein kinase 1 (PDK1) activates many AGC kinases in vitro by phosphorylating the T-loop residue, but whether PDK1 also phosphorylates the hydrophobic motif and whether all other AGC kinases are substrates for PDK1 is unknown. RESULTS: Mouse embryonic stem (ES) cells in which both copies of the PDK1 gene were disrupted were viable. In PDK1(-/-) ES cells, PKB, p70 S6 kinase and p90 Rsk were not activated by stimuli that induced strong activation in PDK1(+/+) cells. Other AGC kinases - namely, protein kinase A (PKA), the mitogen- and stress-activated protein kinase 1 (MSK1) and the AMP-activated protein kinase (AMPK) - had normal activity or were activated normally in PDK1(-/-) cells. The insulin-like growth factor 1 (IGF1) induced PKB phosphorylation at its hydrophobic motif, but not at its T-loop residue, in PDK1(-/-) cells. IGF1 did not induce phosphorylation of p70 S6 kinase at its hydrophobic motif in PDK1(-/-) cells. CONCLUSIONS: PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1(-/-) cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase.     

Regulation of p90RSK phosphorylation by SARS-CoV infection in Vero E6 cells

The 90 kDa ribosomal S6 kinases (p90RSKs) are a family of broadly expressed serine/threonine kinases with two kinase domains activated by extracellular signal-regulated protein kinase in response to many growth factors. Our recent study demonstrated that severe acute respiratory syndrome (SARS)-coronavirus (CoV) infection of monkey kidney Vero E6 cells induces phosphorylation and dephosphorylation of signaling pathways, resulting in apoptosis. In the present study, we investigated the phosphorylation status of p90RSK, which is a well-known substrate of these signaling pathways, in SARS-CoV-infected cells. Vero E6 mainly expressed p90RSK1 and showed weak expression of p90RSK2. In the absence of viral infection, Ser221 in the N-terminal kinase domain was phosphorylated constitutively, whereas both Thr573 in the C-terminal kinase domain and Ser380 between the two kinase domains were not phosphorylated in confluent cells. Ser380, which has been reported to be involved in autophosphorylation by activation of the C-terminal kinase domain, was phosphorylated in confluent SARS-CoV-infected cells, and this phosphorylation was inhibited by , which is an inhibitor of p38 mitogen-activated protein kinases (MAPK). Phosphorylation of Thr573 was not upregulated in SARS-CoV-infected cells. Thus, in virus-infected cells, phosphorylation of Thr573 was not necessary to induce phosphorylation of Ser380. On the other hand, Both Thr573 and Ser380 were phosphorylated by treatment with epidermal growth factor (EGF) in the absence of p38 MAPK activation. Ser220 was constitutively phosphorylated despite infection. These results indicated that phosphorylation status of p90RSK by SARS-CoV infection is different from that by stimulation of EGF. This is the first detailed report regarding regulation of p90RSK phosphorylation by virus infection.     

Differential regulation of mTOR-dependent S6 phosphorylation by muscarinic acetylcholine receptor subtypes

Muscarinic receptors subserve many functions in both peripheral and central nervous systems. Some of these processes depend on increases in protein synthesis, which may be achieved by activation of mammalian target of rapamycin (mTOR), a kinase that regulates protein translation capacity. Here, we examined the regulation of mTOR-dependent signaling pathways by muscarinic receptors in SK-N-SH human neuroblastoma cells, and in human embryonic kidney (HEK) cell lines transfected with individual muscarinic receptor subtypes. In SK-N-SH cells, the acetylcholine analog carbachol stimulated phosphorylation of the ribosomal S6 protein, a downstream target of mTOR. The sensitivity of the response to subtype-selective muscarinic receptor antagonists indicated that it was mediated by M3 receptors. Carbachol-evoked S6 phosphorylation was blocked by the mTOR inhibitor rapamycin, but was independent of phosphoinositide 3-kinase activation. The response was significantly reduced by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also inhibited carbachol-evoked S6 phosphorylation in HEK cells expressing M2 receptors, but was ineffective in M3 receptor-expressing HEK cells, although carbachol activated MAPK in both transfected lines. The p90 ribosomal S6 kinase has been implicated in mTOR regulation by phorbol esters, but was not activated by carbachol in any of the cell lines tested. The protein kinase C inhibitor bisindolylmaleimide I reduced carbachol-stimulated S6 phosphorylation in SK-N-SH cells, and in HEK cells expressing M3 receptors, but not in HEK cells expressing M2 receptors. The results demonstrate that multiple muscarinic receptor subtypes regulate mTOR, and that both MAPK-dependent and -independent mechanisms may mediate the response in a cell context-specific manner.     

Regulation of connexin43 function by activated tyrosine protein kinases

Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. Here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.     

Insulin-activated protein kinases phosphorylate a pseudosubstrate synthetic peptide inhibitor of the p70 S6 kinase

p70 S6 kinase, a major insulin-mitogen-activated ribosomal S6 protein kinase in mammalian cells, is activated by phosphorylation of multiple Ser/Thr residues on the enzyme polypeptide. A synthetic peptide, corresponding to a 37-residue segment from the carboxyl-terminal tail of the kinase which resembles the sequence phosphorylated in S6, acts as a competitive inhibitor of p70 S6 kinase without itself being phosphorylated by the enzyme. This synthetic peptide is phosphorylated by an array of protein kinases which are rapidly activated by insulin. Thus, these sequences of p70 S6 kinase constitute a potential autoinhibitory pseudosubstrate site, whose phosphorylation is catalyzed by candidate upstream-activating protein kinases.     

Pancreastatin,a chromogranin A-derived peptide,activates protein synthesis signaling cascade in rat adipocytes

Pancreastatin (PST), a chromogranin A-derived peptide, has been found to modulate glucose, lipid, and protein metabolism in rat adipocytes. PST has an overall counterregulatory effect on insulin action by activating a specific receptor-effector system (Galpha(q/11) protein-PLC-beta-PKC(classical)). However, PST stimulates both basal and insulin-mediated protein synthesis in rat adipocytes. In order to further investigate the mechanisms underlying the effect of PST stimulating protein synthesis, we sought to study the regulation of different components of the core translational machinery by the signaling triggered by PST. Thus, we studied ribosomal p70 S6 kinase, phosphorylation of the cap-binding protein (initiation factor) eIF4E, and phosphorylation of the eIF4E-binding protein 4E-BP1 (PHAS-I). We have found that PST stimulates the S6 kinase activity, as assessed by kinase assay using specific immunoprecipitates and substrate. This effect was checked by Western blot with specific antibodies against the phosphorylated S6 kinase. Thus, PST dose-dependently stimulates Thr421/Ser424 phosphorylation of S6 kinase. Moreover, PST promotes phosphorylation of regulatory sites in 4E-BP1 (PHAS-I) (Thr37, Thr46). The initiation factor eIF4E itself, whose activity is also increased upon phosphorylation, is phosphorylated in Ser209 by PST stimulation. Finally, we have found that these effects of PST on S6 kinase and the translation machinery can be blocked by preventing the activation of PKC. These results indicate that PST stimulates protein synthesis machinery by activating PKC and provides some evidence of the molecular mechanisms involved, i.e., the activation of S6K and the phosphorylation of 4E-BP1 (PHAS-I) and the initiation factor eIF4E.     

Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D

Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCalpha, PKCbeta1, PKCgamma, and PKCepsilon are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.     

Heat shock protein 90 indirectly regulates ERK activity by affecting Raf protein metabolism

Extracellular signal-regulated protein kinase (ERK) has been implicated in the pathogenesis of several nerve system diseases. As more and more kinases have been discovered to be the client proteins of the molecular chaperone Hsp90, the use of Hsp90 inhibitors to reduce abnormal kinase activity is a new treatment strategy for nerve system diseases. This study investigated the regulation of the ERK pathway by Hsp90. We showed that Hsp90 inhibitors reduce ERK phosphorylation without affecting the total ERK protein level. Further investigation showed that Raf, the UPstream kinase in the Ras-Raf-MEK-ERK pathway, forms a complex with Hsp90 and Hsp70. Treating cells with Hsp90 inhibitors facilitates Raf degradation,thereby down-regulating the activity of ERK.     

Phosphorylation of human microtubule-associated protein tau by protein kinases of the AGC subfamily

Intraneuronal inclusions made of hyperphosphorylated microtubule-associated protein tau are a defining neuropathological characteristic of Alzheimer's disease, and of several other neurodegenerative disorders. Many phosphorylation sites in tau are S/TP sites that flank the microtubule-binding repeats. Others are KXGS motifs in the repeats. One site upstream of the repeats lies in a consensus sequence for AGC kinases. This site (S214) is believed to play an important role in the events leading from normal, soluble to filamentous, insoluble tau. Here, we show that all AGC kinases tested phosphorylated S214. RSK1 and p70 S6 kinase also phosphorylated the neighbouring T212, a TP site that conforms weakly to the AGC kinase consensus sequence. MSK1 phosphorylated S214, as well as S262, a KXGS site in the first repeat, and S305 in the second repeat.     

Production of active recombinant mitogen-activated protein kinases through transient transfection of 293T cells

Mitogen-activated protein (MAP) kinases are a family of serine/threonine protein kinases that play an important role in a myriad of cellular processes, including cell proliferation, differentiation, and apoptosis. Abnormal activation of MAP kinases has been shown to participate in a variety of human diseases which include cancer, septic shock, rheumatoid arthritis, diabetes, and cardiovascular diseases. Active MAP kinase enzymes are not only valuable for basic biomedical research but are also critical for the development of pharmacological inhibitors as therapeutic drugs in the treatment of relevant human diseases. MAP kinases produced in a bacterial system are poorly active due to a lack of proper phosphorylation at their characteristic threonine and tyrosine residues. To overcome these limitations, we have developed a mammalian expression system for high level expression and one-step purification of enzymatically MAP kinases. We cloned JNK1, p38, and p38-regulated MAP kinase-activated protein kinase-2 into the mammalian expression vector pEBG, and expressed these protein kinases as glutathione S-transferase fusion proteins in human embryonic kidney 293T cells through transient transfection. The protein kinases were activated in vivo through treating the transfected cells with sodium arsenite and affinity-purified using glutathione-Sepharose beads. The enzymatic activities of these protein kinases were demonstrated by Western blot analysis and in vitro kinase assays. Our results indicate that this system is an extremely powerful tool for generating valuable reagents, and could be very valuable for proteomic studies.     

Specific induction of heat shock protein 27 by glucocorticoid in osteoblasts

It is generally recognized that osteoporosis is a common complication of patients with glucocorticoid excess and that glucocorticoid receptor is associated with heat shock protein (HSP) 70 and HSP90 in a heterocomplex. In the present study, we investigated whether glucocorticoid induces HSP27, HSP70, and HSP90 in osteoblast-like MC3T3-E1 cells. Dexamethasone time-dependently increased the levels of HSP27, while having no effect on the levels of HSP70 or HSP90. The effect of dexamethasone was dose-dependent in the range between 0.1 nM and 0.1 microM. Dexamethasone induced an increase of the levels of mRNA for HSP27. Dexamethasone induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the HSP27 accumulation by dexamethasone. In addition, SB203580 reduced the dexamethasone-stimulated increase of the mRNA levels for HSP27. The dexamethasone-induced phosphorylation of p38 MAP kinase was reduced by SB203580. These results strongly suggest that glucocorticoid stimulates the induction of neither HSP70 nor HSP90, but HSP27 in osteoblasts, and that p38 MAP kinase is involved in the induction of HSP27.     

Insulin signal transduction through protein kinase cascades

This review summarizes the evolution of ideas concerning insulin signal transduction, the current information on protein ser/thr kinase cascades as signalling intermediates, and their status as participants in insulin regulation of energy metabolism. Best characterized is the Ras-MAPK pathway, whose input is crucial to cell fate decisions, but relatively dispensable in metabolic regulation. By contrast the effectors downstream of PI-3 kinase, although less well elucidated, include elements indispensable for the insulin regulation of glucose transport, glycogen and cAMP metabolism. Considerable information has accrued on PKB/cAkt, a protein kinase that interacts directly with Ptd Ins 3OH phosphorylated lipids, as well as some of the elements further downstream, such as glycogen synthase kinase-3 and the p70 S6 kinase. Finally, some information implicates other erk pathways (e.g. such as the SAPK/JNK pathway) and Nck/cdc42-regulated PAKs (homologs of the yeast Ste 20) as participants in the cellular response to insulin. Thus insulin recruits a broad array of protein (ser/thr) kinases in its target cells to effectuate its characteristic anabolic and anticatabolic programs.