Graham J. Sale

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 p42MAPK/p44MAPKmitogen-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 p70s6k was activated during GH priming, pretreatment of cells with rapamycin, which prevented the activation of p70s6k, 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 p42MAPK/p44MAPKand p70s6k is not essential for the promotion of differentiation by GH, although these signals are required for GH-independent terminal differentiation.

Protein kinase-ζ interacts with munc18c: role in GLUT4 trafficking

Aims/hypothesisInsulin-stimulated glucose transport requires a signalling cascade through kinases protein kinase (PK) Cζ/λ 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 methodsA 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.ResultsThe yeast two-hybrid screen identified PKCζ 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 PKCζ to residues 295–338 and showed that the N-terminal region of PKCζ was required for the interaction. Endogenous munc18c was shown to associate with endogenous PKCζ in vivo in various cell types. Importantly, insulin stimulation increased the association by approximately three-fold. Moreover, disruption of PKCζ binding to munc18c by deletion of residues 295–338 of munc18c or deletion of the N-terminal region of PKCζ markedly inhibited the ability of insulin to stimulate glucose uptake or GLUT4 translocation.Conclusions/interpretationWe have identified a physiological interaction between munc18c and PKCζ that is insulin-regulated. This establishes a link between a kinase (PKCζ) involved in the insulin signalling cascade and a known component of the GLUT4 vesicle trafficking pathway (munc18c). The results indicate that PKCζ regulates munc18c and suggest a model whereby insulin triggers the docking of PKCζ to munc18c, resulting in enhanced GLUT4 translocation to the plasma membrane.

Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking

PKCzeta (protein kinase Czeta) is a serine/threonine protein kinase controlled by insulin, various growth factors and phosphoinositide 3-kinase. It has been implicated in controlling glucose transport in response to insulin by the translocation of GLUT4-(glucose transporter 4) containing vesicles to the plasma membrane in stimulated cells. How PKCzeta modulates GLUT4 vesicle trafficking remains unknown. A yeast two-hybrid screen using full-length human PKCzeta identified 80K-H protein as an interactor with PKCzeta. GST (glutathione S-transferase) pull-down assays with GST-tagged 80K-H constructs confirmed the interaction and showed that the N-terminal portion of 80K-H was not required for the interaction. Immunoprecipitates of endogenous PKCzeta from Cho cells, 3T3-L1 adipocytes or L6 myotubes contained endogenous 80K-H, demonstrating a physiological interaction. Insulin stimulation enhanced the association 3-5-fold. Immunoprecipitates of endogenous 80K-H contained endogenous munc18c and immunoprecipitates of endogenous munc18c contained endogenous PKCzeta, with insulin markedly increasing the amount of co-immunoprecipitated protein in each case. These results show that insulin triggers interactions in vivo between PKCzeta, 80K-H and munc18c. Overexpression of 80K-H constructs mimicked the action of insulin in stimulating both glucose uptake and translocation of Myc-tagged GLUT4 in Cho cells, with the level of effect proportional to the ability of the constructs to associate with munc18c. These results identify 80K-H as a new player involved in GLUT4 vesicle transport and identify a link between a kinase involved in the insulin signalling cascade, PKCzeta, and a known component of the GLUT4 vesicle trafficking pathway, munc18c. The results suggest a model whereby insulin triggers the formation of a PKCzeta-80K-H-munc18c complex that enhances GLUT4 translocation to the plasma membrane.

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

The protein-tyrosine phosphatase PTPα 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 PTPα, a potent phosphorothioate oligonucleotide-based antisense strategy was developed that specifically depleted endogenous PTPα from 3T3-L1 adipocytes. The antisense probe, αAS1, achieved PTPα depletion levels normally of ≥85% and which varied up to levels where PTPα was not detected at all. Elimination of PTPα by 85% inhibited c-Src activity by 80%. Abolishing PTPα 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 αAS1. It is concluded that endogenous PTPα is a key regulator of c-Src activity in 3T3-L1 adipocytes and that PTPα 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, αAS1, provides an important molecular tool of general applicability for further dissecting the roles and precise targets of endogenous PTPα.

Knock-down of LAR protein tyrosine phosphatase induces insulin resistance

To test the role of the leukocyte common antigen‐related protein tyrosine phosphatase (LAR) as a regulator of insulin receptor (IR) signalling, an siRNA probe against LAR was developed. Knock‐down of LAR induced post‐receptor insulin resistance with the insulin‐induced activation of PKB/Akt and MAP kinases markedly inhibited. The phosphorylation and dephosphorylation of the IR and insulin receptor substrate (IRS) proteins were unaffected by LAR knock‐down. These results identify LAR as a crucial regulator of the sensitivity of two key insulin signalling pathways to insulin. Moreover, the siRNA probe provides a molecular tool of general applicability for further dissecting the precise targets and roles of LAR.

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.

Assay of phosphotyrosyl protein phosphatase using synthetic peptide 1142–1153 of the insulin receptor

Synthetic peptide 1142–1153 of the insulin receptor was phosphorylated on tyrosine by the insulin receptor and found to be a potent substrate for dephosphorylation by rat liver particulate and soluble phosphotyrosyl protein phosphatases. Apparent K m values were 5 μM. V m values (nmol phosphate removed/min per mg protein) were 0.62 (particulate) and 0.2 (soluble). This corresponds to 80% of total activity being membrane‐associated, indicating that membrane‐bound phosphatases are important receptor phosphatases. The phosphatase activities were distinct from acid and alkaline phosphatase. In conclusion peptide 1142–1153 provides a useful tool for the further study and characterization of phosphotyrosyl protein phosphatases.

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.

Identification of the active site polypeptide in labeled photoreceptor membranes digested with papain.

It is now generally recognized that an understanding of the’mechanism through which photopic stimulation is translated into a visual impulse requires a knowledge of the organization of rhodopsin in the disc membrane of rod outer segments (ROS). Attempts directed towards this end include the work of Trayhurn et al. [ 1,2] who observed that rhodopsin in disc membranes was digested by papain into a functionally active membrane bound fragment, termed rhodopsin-core, which was assigned a mol.wt of 24 800 based on sodium dodecyl sulphate (SDS)acrylamide gel electrophoresis. It was inferred that the retinal binding site resided in the rhodopsin-core and that in its formation about one-third of the polypeptide chain had been removed from rhodopsin (mol.wt 36 000). Using ROS in which rhodopsin is specifically labelled with tritium at the active site we have now critically examined the location of the retinal binding site in ROS digested with papain. Our results show that papain cleaves rhodopsin in disc membranes into fragments held as a non-covalent complex in which a polypeptide of mol.wt 15 500 contains the retinal binding site. From the evidence presented we have concluded that papain digestion of rhodopsin in disc membranes does not produce a true rhodopsin-core, but a multiple-cleaved species hereafter referred to as cleaved-rhodopsin.

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.