Protein kinase B: signalling roles and therapeutic targeting.

The serine/threonine kinase, protein kinase B (PKB, also known as Akt), is activated by a wide array of growth factors and insulin. PKB is a central player in the regulation of metabolism, apoptosis, transcription and the cell-cycle. PKB exists as three isoforms (alpha, beta and gamma) that may have unique as well as common functions within the cell. Deregulation of PKB is associated with several human diseases, including cancer, diabetes and schizophrenia. These findings underscore the medical relevance of the PKB pathway and make PKB an attractive drug target for the treatment of diseases that exhibit abnormal PKB signalling.

Protein kinase-zeta interacts with munc18c: role in GLUT4 trafficking.

AIMS/HYPOTHESIS: Insulin-stimulated glucose transport requires a signalling cascade through kinases protein kinase (PK) Czeta/lambda and PKB that leads to movement of GLUT4 vesicles to the plasma membrane. The aim of this study was to identify missing links between the upstream insulin-regulated kinases and the GLUT4 vesicle trafficking system. MATERIALS AND METHODS: A yeast two-hybrid screen was conducted, using as bait full-length mouse munc18c, a protein known to be part of the GLUT4 vesicle trafficking machinery. RESULTS: The yeast two-hybrid screen identified PKCzeta as a novel interactor with munc18c. Glutathione S transferase (GST) pull-downs with GST-tagged munc18c constructs confirmed the interaction, mapped a key region of munc18c that binds PKCzeta to residues 295-338 and showed that the N-terminal region of PKCzeta was required for the interaction. Endogenous munc18c was shown to associate with endogenous PKCzeta in vivo in various cell types. Importantly, insulin stimulation increased the association by approximately three-fold. Moreover, disruption of PKCzeta binding to munc18c by deletion of residues 295-338 of munc18c or deletion of the N-terminal region of PKCzeta markedly inhibited the ability of insulin to stimulate glucose uptake or GLUT4 translocation. CONCLUSIONS/INTERPRETATION: We have identified a physiological interaction between munc18c and PKCzeta that is insulin-regulated. This establishes a link between a kinase (PKCzeta) involved in the insulin signalling cascade and a known component of the GLUT4 vesicle trafficking pathway (munc18c). The results indicate that PKCzeta regulates munc18c and suggest a model whereby insulin triggers the docking of PKCzeta to munc18c, resulting in enhanced GLUT4 translocation to the plasma membrane.

Downregulation of the ERK 1 and 2 mitogen activated protein kinases using antisense oligonucleotides inhibits proliferation of porcine vascular smooth muscle cells.

The current model of the arterial response to injury suggests that proliferation of vascular smooth muscle cells is a central event. Mitogen activated protein kinases are part of the final common pathway of intracellular signalling involved in cell division and thus constitute an attractive target in attempting to inhibit this proliferation. We hypothesised that antisense oligonucleotides to mitogen activated protein kinase would inhibit serum induced smooth muscle cell proliferation by downregulating the protein. Porcine vascular smooth muscle cells were cultured and an antisense oligonucleotide sequence against the ERK family of mitogen activated protein kinases (AMK1) was introduced by liposomal transfection. Sense oligonucleotides and a random sequence were used as controls. Proliferation was inhibited by AMK1 versus the sense controls, as assessed by tritiated thymidine incorporation (P<0.01). Immunoblots revealed downregulation of the target protein by AMK1 by 63% versus the sense control (P<0.05). In conclusion, antisense oligonucleotides specifically inhibited proliferation and downregulated the target protein. This is consistent with a central role for mitogen activated protein kinases in vascular smooth muscle cell proliferation in the porcine model. In addition, the data suggest a possible role for antisense oligonucleotides in the modulation of the arterial injury response.

Use of an antisense strategy to dissect the signaling role of protein-tyrosine phosphatase alpha.

The protein-tyrosine phosphatase PTPalpha has been proposed to play an important role in controlling the dephosphorylation of a number of key signaling proteins and in regulating insulin signaling. To examine the potential cellular functions and physiological substrates of PTPalpha, a potent phosphorothioate oligonucleotide-based antisense strategy was developed that specifically depleted endogenous PTPalpha from 3T3-L1 adipocytes. The antisense probe, alphaAS1, achieved PTPalpha depletion levels normally of >/=85% and which varied up to levels where PTPalpha was not detected at all. Elimination of PTPalpha by 85% inhibited c-Src activity by 80%. Abolishing PTPalpha to levels undetected did not alter the tyrosine dephosphorylation of the insulin receptor or insulin receptor substrate proteins. Moreover, the ability of insulin to activate ERK2 or to stimulate DNA synthesis was not altered by alphaAS1. It is concluded that endogenous PTPalpha is a key regulator of c-Src activity in 3T3-L1 adipocytes and that PTPalpha is not required for the dephosphorylation of the insulin receptor or the insulin receptor substrate proteins or for the regulation of several downstream insulin signaling events in 3T3-L1 adipocytes. Finally, the development of the antisense probe, alphaAS1, provides an important molecular tool of general applicability for further dissecting the roles and precise targets of endogenous PTPalpha.

Role of ERK1/ERK2 and p70S6K pathway in insulin signalling of protein synthesis.

The signalling pathways by which insulin triggers protein synthesis were studied using an antisense strategy to deplete ERK1/ERK2 and rapamycin to inhibit the p70S6K pathway. The results indicated that ERK1/ERK2 principally regulated the amount of the protein synthesis machinery available in the cell while the p70S6K pathway contributed to modulating its activation in response to insulin. ERK1/ERK2 also mediated in a small proportion of insulin-stimulated protein synthesis which included the induction of c-fos protein. When c-fos induction was blocked the majority of insulin-stimulated protein synthesis still occurred and thus did not require transcriptional regulation of c-fos or its targets.

Growth hormone-dependent differentiation of 3T3-F442A preadipocytes requires Janus kinase/signal transducer and activator of transcription but not mitogen-activated protein kinase or p70 S6 kinase signaling.

The signals mediating growth hormone (GH)-dependent differentiation of 3T3-F442A preadipocytes under serum-free conditions have been studied. GH priming of cells was required before the induction of terminal differentiation by a combination of epidermal growth factor, tri-iodothyronine, and insulin. Cellular depletion of Janus kinase-2 (JAK-2) using antisense oligodeoxynucleotides (ODNs) prevented GH-stimulated JAK-2 and signal transducer and activator of transcription (STAT)-5 tyrosine phosphorylation and severely attenuated the ability of GH to promote differentiation. Although p42(MAPK)/p44(MAPK) mitogen-activated protein kinases were activated during GH priming, treatment of cells with PD 098059, which prevented activation of these kinases, did not block GH priming. However, antisense ODN-mediated depletion of mitogen-activated protein kinases from the cells showed that their expression was necessary for terminal differentiation. Similarly, although p70(s6k) was activated during GH priming, pretreatment of cells with rapamycin, which prevented the activation of p70(s6k), had no effect on GH priming. However, rapamycin did partially block epidermal growth factor, tri-iodothyronine, and insulin-stimulated terminal differentiation. By contrast, cellular depletion of STAT-5 with antisense ODNs completely abolished the ability of GH to promote differentiation. These results indicate that JAK-2, acting specifically via STAT-5, is necessary for GH-dependent differentiation of 3T3-F442A preadipocytes. Activation of p42(MAPK)/p44(MAPK) and p70(s6k) is not essential for the promotion of differentiation by GH, although these signals are required for GH-independent terminal differentiation.

PHAS-I phosphorylation in response to foetal bovine serum (FBS) is regulated by an ERK1/ERK2-independent and rapamycin-sensitive pathway in 3T3-L1 adipocytes.

The phosphorylation state of PHAS-I is thought to be important in the regulation of protein synthesis initiation. PHAS-I phosphorylation significantly increases in response to growth factors and insulin. ERK1/ERK2 have previously been implicated as PHAS-I kinases. Present work utilised a specific phosphorothioate oligonucleotide antisense strategy against ERK1/ERK2 to determine whether ERK1/ERK2 mediate FBS-stimulated PHAS-I phosphorylation in vivo. Depleting > 90% of cellular ERK1/ERK2 had no effect on FBS-stimulated PHAS-I phosphorylation. However, treatment of cells with a specific p70S6k pathway inhibitor, rapamycin, markedly attenuated FBS-stimulated PHAS-I phosphorylation. These results indicate that PHAS-I phosphorylation in response to FBS occurs through an ERK1/ERK2-independent and rapamycin-sensitive pathway in 3T3-L1 adipocytes.

Purification and characterization of an insulin-stimulated insulin receptor serine kinase.

In cells, insulin stimulates autophosphorylation of the insulin receptor on tyrosine and its phosphorylation on serine and threonine by poorly characterized kinases. Here we describe methods for the purification of an insulin-stimulated insulin receptor serine kinase from human placenta and rat liver by sequential chromatography of solubilized membranes on wheat germ agglutinin-agarose, Mono Q, phenyl-Superose, and Superose 12. On silver-stained SDS-polyacrylamide gels, the resulting kinase was homogeneous (human) or near-homogeneous (rat) and had an apparent M(r) of 40000. The apparent M(r) determined by gel filtration was also 40000, suggesting that the kinase exists as a monomer. The kinase could be reconstituted back to the insulin receptor stripped of the kinase to yield a high stoichiometry of serine phosphorylation of the insulin receptor in the presence of insulin (0.75 +/- 0.15 mol/mol of beta-subunit, mean +/- SEM, n = 3). The activity of the reconstituted kinase toward the insulin receptor was insulin-regulated, being stimulated > 5-fold by insulin. Insulin increased the catalytic activity of the reconstituted kinase. The purified kinase specifically phosphorylated serine 1078 of the insulin receptor, a major site of insulin-stimulated serine phosphorylation in vivo, showing that the purified kinase phosphorylated a physiologically relevant site on the insulin receptor. Phosphorylation of serine 1078 of the insulin receptor to high stoichiometry by the kinase did not affect insulin-stimulated exogenous protein tyrosine kinase activity of the insulin receptor. Similarly, insulin receptor phosphorylated with or without the purified kinase exhibited the same levels of tyrosine autophosphorylation and of the tyrosine kinase-activating tris-phosphorylated kinase domain species. Properties of the kinase distinguished it from kinases known to act on the insulin receptor and other kinases that are insulin-stimulated, indicating that the kinase is a novel entity. The serine kinase underwent autophosphorylation on serine and immunoprecipitated with the insulin receptor. The availability of the purified kinase should facilitate cloning of the kinase, determination of the mechanism of activation of the kinase, and study of the wider potential role of the kinase in insulin signalling, and the ability to be able to phosphorylate serine 1078 to high stoichiometry should facilitate further studies into the function of this serine phosphorylation site.

Depletion of mitogen-activated protein kinase using an antisense oligodeoxynucleotide approach downregulates the phenylephrine-induced hypertrophic response in rat cardiac myocytes.

An antisense oligodeoxynucleotide (ODN) approach was used to investigate whether mitogen-activated protein kinase (MAPK) is necessary for the hypertrophic response in cardiac myocytes. A phosphorothioate-protected 17-mer directed against the initiation of translation sites of the p42 and p44 MAPK isoform mRNAs was introduced into cultured cardiac myocytes by liposomal transfection. At an antisense ODN concentration of 0.2 mumol/L, p42 MAPK protein was reduced by 82% (immunoblot) after 48 hours, and p42 and p44 MAPK activities were reduced by 44% and 60%, respectively. The same concentration of anti-MAPK ODN inhibited development of the morphological features of hypertrophy (sarcomerogenesis, increased cell size) in myocytes exposed to phenylephrine. Phenylephrine-induced activation of the atrial natriuretic factor (ANF) promoter (measured by the activity of a transfected ANF promoter/luciferase reporter gene) and induction of ANF mRNA (measured by RNase protection assay) were also attenuated. We conclude that MAPK is important for the development of the hypertrophic phenotype in this model of hypertrophy.

Studies into the identity of the sites of insulin-stimulated insulin receptor serine phosphorylation. Characterization of synthetic peptide substrates for the insulin-stimulated insulin receptor serine kinase.

The identity of the sites of insulin-stimulated serine phosphorylation in the human insulin receptor was examined by synthesizing peptides that together encompassed all the serine residues of the cytosolic portion of the beta-subunit and testing them as substrates for phosphorylation by a preparation of human insulin receptor copurified with insulin-stimulated insulin receptor serine kinase activity. Of the 14 peptides studied, only 4 (1071--1080, 1290--1298, 1253--1271, and 1313--1329) were phosphorylated on serine, with the serine phosphorylation stimulated 2--4-fold by insulin. Peptides 1071--1080 and 1290--1298 were 3--7-fold better substrates for the serine phosphorylation than the other serine-phosphorylated peptides. Peptides 1071--1080 and 1313--1329 also exhibited insulin-stimulated phosphorylation on tyrosine. Two-dimensional thin-layer tryptic mapping of the phosphorylated insulin receptor/insulin-stimulated insulin receptor serine kinase preparation or of insulin receptor phosphorylated in human Hep G2 cells yielded two major peptides, called S1 and S2, that ran as a pair of closely migrating spots, and other lesser peptides that contained phosphoserine. S1 and S2 also contained some phosphotyrosine and gave phosposerine/phosphotyrosine ratios of approximately 6 and 0.96-1.50 for the in vivo and in vitro labeled receptor, respectively. S1 and S2 were not cleaved by V8. Of the serine-phosphorylated peptides, only 1290--1298 and 1071--1080 should be V8 resistant; 1290--1298 contains serine sites 1293/4 and migrated distinctly from S1 and S2 in tryptic maps. Peptide 1071--1080 mimicked the production of S1 and S2 in tryptic maps yielding a doublet of phosphopeptides, each containing phosphoserine and phosphotyrosine, which comigrated exactly with S1 and S2. Comigration was confirmed at a different pH and by mixing experiments. Radiosequenation showed that serine 1078 was phosphorylated. Tyrosine 1075 was also phosphorylated, but it was no more than a minor site in vivo. It is concluded that serine 1078 of the insulin receptor is a major site of insulin-stimulated phosphorylation in vivo and in vitro. The peptide sequences provide a range of substrates to facilitate the study, purification, and characterization of the insulin-stimulated insulin receptor serine kinase or kinases, and the identification of a major site of insulin-stimulated serine phosphorylation will help elucidate the function of the insulin receptor serine phosphorylation.

Studies on an insulin-stimulated insulin receptor serine kinase activity: separation of the kinase activity from the insulin receptor and its reconstitution back to the insulin receptor.

In cells insulin stimulates autophosphorylation of the insulin receptor on tyrosine and its phosphorylation on serine and threonine by poorly characterized kinases. Recently we have achieved co-purification of the insulin receptor with insulin-stimulated insulin receptor serine kinase activity. We now show that the co-purified serine kinase activity can be removed by NaCl washing and reconstituted by adding back the NaCl eluate. Reconstitution enabled higher serine phosphorylation than achieved with the co-purified preparation. Myelin basic protein was discovered to be a potent substrate for insulin-stimulated serine phosphorylation by the co-purified preparation, with the activity responsible having similar properties to the serine kinase activity towards the receptor. Myelin basic protein was also phosphorylated on serine by the NaCl eluate. Myelin basic protein phosphorylated by the co-purified preparation or the NaCl eluate gave the same set of phosphoserine peptides. The major myelin basic protein serine kinase activity in the NaCl eluate co-purified exactly on Mono Q with the activity that restored insulin-stimulated insulin receptor serine phosphorylation. These results provide strong evidence for the true separation of the serine kinase from the insulin receptor and the distinctiveness of the serine kinase activity from the insulin receptor tyrosine kinase and mitogen-activated protein kinases. The procedures developed for the isolation of the serine kinase and the establishment of an effective in vitro substrate should allow purification of the kinase. The protocols also provide flexible systems for identifying the functions of the insulin-stimulated serine phosphorylations and the respective kinase(s).

Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis.

A phosphorothioate-oligonucleotide-based antisense strategy for depleting MAP kinase was developed. The 17mer antisense probe, EAS 1, caused a potent and concentration-dependent decrease in the steady state expression of p42 and p44 MAP kinase in 3T3 L1 fibroblasts and adipocytes with submicromolar concentrations effective. Antisense EAS 1 elicited a dose-dependent inhibition of insulin- and serum-stimulated DNA synthesis. Elimination of p42 MAP kinase by > 95% and p44 MAP kinase to levels undetected blocked the ability of serum in 3T3 L1 fibroblasts and insulin in 3T3 L1 adipocytes to stimulate DNA synthesis by 87-95%. The differentiation of 3T3 L1 fibroblasts into adipocytes was prevented by 1 microM antisense EAS 1. The corresponding sense, scrambled or sense plus antisense EAS 1 phosphorothioate oligonucleotides did not deplete the p42 or p44 MAP kinase from either cell type, did not inhibit stimulation of DNA synthesis and did not interfere with differentiation. Two kinases on different MAP kinase activation pathways were not depleted by antisense EAS 1 whereas the ability of insulin to activate p90 S6 kinase was > 90% eliminated in 3T3 L1 adipocytes by 4.5 microM antisense EAS 1. In conclusion these results show that MAP kinase is required for insulin and serum stimulation of DNA synthesis, for insulin stimulation of p90 S6 kinase activity and for differentiation of 3T3 L1 cells. Moreover, the development of the antisense probe EAS 1 against a target sequence of p42 MAP kinase that is conserved in p44 MAP kinase and across a range of species provides a molecular tool of general applicability for further dissecting the precise targets and roles of MAP kinase.

Regulation of an hepatic low-M(r) membrane-associated protein-tyrosine phosphatase.

Protein-tyrosine phosphatases (PTPases), active against autophosphorylated insulin and epidermal growth factor (EGF) receptors in rat liver, are predominantly membrane associated. Fasting of rats for 48 h decreased hepatic particulate PTPase activity by 15.0-26.9%. This reduction in particulate PTPase activity was due to a rather specific decrease in activity of > 85% of a single species of PTPase, termed PTPase I. Disappearance of PTPase I activity from the particulate fraction was not accounted for by its translocation to the cytosol. PTPase I displayed the highest activity against autophosphorylated insulin and EGF receptors, relative to activity against a 32P-labelled peptide substrate, of three PTPases resolved from the liver particulate fraction. The M(r) value of PTPase I, as determined by gel filtration on a Superose 12 column was approx. 42,000, indicating that PTPase I belongs to the low-M(r) class of PTPases. An antibody raised against PTPase 1B, the prototype of this class of PTPases, did not react with PTPase I in Western blots. The potential importance of the novel change in activity of PTPase I in the regulation of insulin-receptor signal transduction is discussed.

Dephosphorylation of autophosphorylated insulin and epidermal-growth-factor receptors by two major subtypes of protein-tyrosine-phosphatase from human placenta.

The identity of protein-tyrosine-phosphatases (PTPases) active against autophosphorylated insulin receptor was probed by using an insulin-receptor-related peptide phosphorylated on tyrosine (peptide 1142-1153). Two major peaks of PTPase activity were resolved from the particulate (Triton X-100-soluble) fraction of human placenta by chromatography on DEAE-cellulose. The two peaks were purified 1300-2300-fold; other peaks of PTPase activity (greater than 15%) were not detected. Properties of the PTPases indicated that they corresponded to subtypes 1A and 1B. Both subtypes appeared capable of catalysing dephosphorylation of all autophosphorylation sites in three domains of the insulin receptor, with no appreciable difference in the pattern of dephosphorylation detected by two-dimensional tryptic-peptide mapping. The tyrosine-1150 domain of the insulin receptor in triply phosphorylated form was found to be highly sensitive to the action of both PTPases, and was dephosphorylated at least 4 times faster than the doubly and singly phosphorylated forms of the tyrosine-1150 domain or phosphorylation sites in other domains by either PTPase. This is significant, as the level of the triphosphotyrosine-1150 species has been shown to correlate well with the capacity of the insulin-receptor tyrosine kinase to phosphorylate other proteins. Both subtypes also dephosphorylated autophosphorylated epidermal-growth-factor (EGF) receptor by greater than 95%. Placental particulate (and cytosolic) PTPase activity against either receptor distributed approximately 2:1 between subtypes 1A and 1B as assayed in the presence of EDTA. In summary, PTPases within two major subtypes have been identified as phosphotyrosyl-insulin and -EGF-receptor phosphatases in vitro. The PTPases identified exhibit high affinities for substrates and high activities in cells, which is commensurate with the PTPases being important in vivo in controlling or reversing autophosphorylation-induced regulatory or signalling events.

Site-specific dephosphorylation and deactivation of the human insulin receptor tyrosine kinase by particulate and soluble phosphotyrosyl protein phosphatases.

Insulin receptor tyrosine kinase activation, induced by insulin-stimulated autophosphorylation, was measured using a synthetic peptide containing residues 1142-1153 of the insulin receptor and shown to be reversed by both particulate and soluble phosphotyrosyl protein phosphatases from rat liver. Deactivation of the tyrosine kinase was highly sensitive to phosphatase action and was correlated best with disappearance of insulin receptors triphosphorylated in the tyrosine-1150 domain. Dephosphorylation of the di- and mono-phosphorylated forms of the tyrosine-1150 domain generated during dephosphorylation or of phosphorylation sites in the C-terminal or putative juxta-membrane domains occurred 3- greater than 10-fold more slowly than deactivation of the tyrosine kinase, and these phosphorylated species did not appear to appreciably (less than 20%) contribute to tyrosine kinase activation. These results indicate that the transition from the triply to the doubly phosphorylated form of the tyrosine-1150 domain acts as an important switch for deactivation of the insulin receptor tyrosine kinase during dephosphorylation. The exquisite sensitivity of this dephosphorylation/deactivation event to phosphotyrosyl protein phosphatase action, combined with the high affinities of this phosphatases for substrates and the high activities of the phosphatases in cells, suggests that the tyrosine kinase activity expressed by insulin-stimulated insulin receptors is likely to be stringently regulated.

Dephosphorylation of insulin-receptor autophosphorylation sites by particulate and soluble phosphotyrosyl-protein phosphatases.

Insulin stimulates autophosphorylation of the insulin receptor on multiple tyrosines in three domains: tyrosines 1316 and 1322 in the C-terminal tail, 1146, 1150 and 1151 in the tyrosine-1150 domain, and possibly 953, 960 or 972 in the juxtamembrane domain. In the present work the sequence of dephosphorylation of the various autophosphorylation sites by particulate and cytosolic preparations of phosphotyrosyl-protein phosphatase from rat liver was studied with autophosphorylated human placental insulin receptor as substrate. Both phosphatase preparations elicited a broadly similar pattern of dephosphorylation. The tyrosine-1150 domain in triphosphorylated form was found to be exquisitely sensitive to dephosphorylation, and was dephosphorylated 3-10-fold faster than the di- and monophosphorylated forms of the tyrosine-1150 domain or phosphorylation sites in other domains. The major route for dephosphorylation of the triphosphorylated tyrosine-1150 domain involved dephosphorylation of one of the phosphotyrosyl pair, 1150/1151, followed by phosphotyrosyl 1146 to generate a species monophosphorylated mainly (greater than 80%) at tyrosine 1150 or 1151. Insulin receptors monophosphorylated in the tyrosine-1150 domain disappeared slowly, and overall the other domains were completely dephosphorylated faster than the tyrosine-1150 domain. Dephosphorylation of the diphosphorylated C-terminal domain yielded insulin receptor in which the domain was singly phosphorylated at tyrosine 1322. Triphosphorylation of the insulin receptor in the tyrosine-1150 domain appears important in activating the receptor tyrosine kinase to phosphorylate other proteins. The extreme sensitivity of the triphosphorylated form of the tyrosine-1150 domain to dephosphorylation may thus be important in terminating or regulating insulin-receptor tyrosine kinase action and insulin signalling.

Characterization of sites of serine phosphorylation in human placental insulin receptor copurified with insulin-stimulated serine kinase activity by two-dimensional thin-layer peptide mapping.

Insulin receptor was copurified from human placenta together with insulin-stimulated kinase activity that phosphorylates the insulin receptor on serine residues. Analysis of phosphorylated insulin receptor by two-dimensional tryptic peptide mapping showed that sites of insulin stimulated serine phosphorylation in the insulin receptor were recovered in the same peptides as those known to be phosphorylated on serine in vivo in response to insulin. This indicates that the serine kinase copurified with the insulin receptor represents a physiologically important enzyme involved in the insulin triggered serine phosphorylation of the insulin receptor in vivo.