Inhibition of insulin signaling in endothelial cells by protein kinase C-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase (PI3K)

The regulation of endothelial function by insulin is consistently abnormal in insulin-resistant states and diabetes. Protein kinase C (PKC) activation has been reported to inhibit insulin signaling selectively in endothelial cells via the insulin receptor substrate/PI3K/Akt pathway to reduce the activation of endothelial nitric-oxide synthase (eNOS). In this study, it was observed that PKC activation differentially inhibited insulin receptor substrate 1/2 (IRS1/2) signaling of insulin's activation of PI3K/eNOS by decreasing only tyrosine phosphorylation of IRS2. In addition, PKC activation, by general activator and specifically by angiotensin II, increased the phosphorylation of p85/PI3K, which decreases its association with IRS1 and activation. Thr-86 of p85/PI3K was identified to be phosphorylated by PKC activation and confirmed to affect IRS1-mediated activation of Akt/eNOS by insulin and VEGF using a deletion mutant of the Thr-86 region of p85/PI3K. Thus, PKC and angiotensin-induced phosphorylation of Thr-86 of p85/PI3K may partially inhibit the activation of PI3K/eNOS by multiple cytokines and contribute to endothelial dysfunction in metabolic disorders.     

Na/H exchange regulatory factor 1, a novel AKT-associating protein, regulates extracellular signal-regulated kinase signaling through a B-Raf-mediated pathway

Na/H exchange regulatory factor 1 (NHERF1) is a scaffolding protein that regulates signaling and trafficking of several G protein-coupled receptors (GPCRs), including the parathyroid hormone receptor (PTH1R). GPCRs activate extracellular signal-regulated kinase (ERK)1/2 through different mechanisms. Here, we characterized NHERF1 regulation of PTH1R-stimulated ERK1/2. Parathyroid hormone (PTH) stimulated ERK1/2 phosphorylation by a protein kinase A (PKA)-dependent, but protein kinase C-, cyclic adenosine 5'-monophosphate-, and Rap1-independent pathway in Chinese hamster ovary cells stably transfected with the PTH1R and engineered to express NHERF1 under the control of tetracycline. NHERF1 blocked PTH-induced ERK1/2 phosphorylation downstream of PKA. This suggested that NHERF1 inhibitory effects on ERK1/2 occur at a postreceptor locus. Forskolin activated ERK1/2, and this effect was blocked by NHERF1. NHERF1 interacted with AKT and inhibited ERK1/2 activation by decreasing the stimulatory effect of 14-3-3 binding to B-Raf, while increasing the inhibitory influence of AKT negative regulation on ERK1/2 activation. This novel regulatory mechanism provides a new model by which cytoplasmic adapter proteins modulate ERK1/2 activation through a receptor-independent mechanism involving B-Raf.     

Cellular adhesion of primary Sertoli cells affects responsiveness of the extracellular signal-regulated kinases 1 and 2 to follicle-stimulating hormone but not to epidermal growth factor

The cellular adhesion status and the exposure to soluble growth factors both contribute to mitogen-activated protein (MAP) kinase activation. To date, however, whether mitogens acting through G-protein-coupled receptors (GPCRs) need cell adhesion to activate MAP kinases/extracellular signal-regulated kinases (ERK) 1, 2 has been poorly investigated. We addressed this point in primary cultures of Sertoli cells experimentally maintained in suspension, considering that follicle-stimulating hormone (FSH) activates ERK1, 2 in attached Sertoli cells by acting through a GPCR. We found that FSH actively repressed ERK1, 2, in a cAMP-dependent but cAMP-dependent protein kinase (PKA)-independent manner, and this inhibition required the activity of a tyrosine phosphatase. In comparison, in the absence of anchorage, ERK1, 2 were still activated by epidermal growth factor, in a PKA-dependent manner. Altogether, these data suggest that sensitivity of the MAP kinase response toward cell adhesion may depend, at least in part, on the class of receptor, GPCR or receptor with tyrosine kinase activity, by which it is triggered.     

Requirement for PI 3-kinase gamma in macrophage migration to MCP-1 and CSF-1

Phosphoinositide 3-kinases (PI3Ks) are important regulators of cell migration. The PI3K isoform gamma is primarily expressed in haematopoietic cells, and is activated by G protein-coupled receptors (GPCRs). Here, we investigate the contribution of PI3Kgamma to macrophage responses to chemoattractants, using bone marrow-derived macrophages from wild-type and PI3Kgamma-null mice. We observe that early membrane ruffling induced by MCP-1, which activates a GPCR, or by CSF-1, which activates a tyrosine kinase receptor, is unaltered in PI3Kgamma(-/-) mice, although by 30 min MCP-1-induced cell polarization was strongly reduced in PI3Kgamma(-/-) compared to wild-type macrophages. The migration behaviour of the macrophages was analysed by time-lapse microscopy in Dunn chemotaxis chambers. PI3Kgamma(-/-) macrophages showed reduced migration speed and translocation, and no chemotaxis to MCP-1. Interestingly, there was also a reduction in migration efficiency in PI3Kgamma(-/-) macrophages stimulated with CSF-1 although early CSF-1R signalling was normal. These results indicate that the initial actin reorganization induced by either a GPCR or tyrosine kinase receptor agonist is not dependent on PI3Kgamma, whereas PI3Kgamma is needed for optimal migration of macrophages to either agonist.     

Alpha-synuclein overexpression negatively regulates insulin receptor substrate 1 by activating mTORC1/S6K1 signaling

Alpha-synuclein (α-Syn) is a major component of Lewy bodies, a pathological feature of Parkinson's and other neurodegenerative diseases collectively known as synucleinopathies. Among the possible mechanisms of α-Syn-mediated neurotoxicity is interference with cytoprotective pathways such as insulin signaling. Insulin receptor substrate (IRS)-1 is a docking protein linking IRs to downstream signaling pathways such as phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K)1; the latter exerts negative feedback control on insulin signaling, which is impaired in Alzheimer's disease. Our previous study found that α-Syn overexpression can inhibit protein phosphatase (PP)2A activity, which is involved in the protective mechanism of insulin signaling. In this study, we found an increase in IRS-1 phosphorylation at Ser636 and decrease in tyrosine phosphorylation, which accelerated IRS-1 turnover and reduced insulin-Akt signaling in α-Syn-overexpressing SK-N-SH cells and transgenic mice. The mTOR complex (C)1/S6K1 blocker rapamycin inhibited the phosphorylation of IRS-1 at Ser636 in cells overexpressing α-Syn, suggesting that mTORC1/S6K1 activation by α-Syn causes feedback inhibition of insulin signaling via suppression of IRS-1 function. α-Syn overexpression also inhibited PP2A activity, while the PP2A agonist C2 ceramide suppressed both S6K1 activation and IRS-1 Ser636 phosphorylation upon α-Syn overexpression. Thus, α-Syn overexpression negatively regulated IRS-1 via mTORC1/S6K1 signaling while activation of PP2A reverses this process. These results provide evidence for a link between α-Syn and IRS-1 that may represent a novel mechanism for α-Syn-associated pathogenesis.     

Protein kinase A‐mediated phosphorylation of naked cuticle homolog 2 stimulates cell‐surface delivery of transforming growth factor‐α for epidermal growth factor receptor transactivation

The classic mode of G protein‐coupled receptor (GPCR)‐mediated transactivation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) transactivation occurs via matrix metalloprotease (MMP)‐mediated cleavage of plasma membrane‐anchored EGFR ligands. Herein, we show that the Gαs‐activating GPCR ligands vasoactive intestinal peptide (VIP) and prostaglandin E₂ (PGE₂) transactivate EGFR through increased cell‐surface delivery of the EGFR ligand transforming growth factor‐α (TGFα) in polarizing madin‐darby canine kidney (MDCK) and Caco‐2 cells. This is achieved by PKA‐mediated phosphorylation of naked cuticle homolog 2 (NKD2), previously shown to bind TGFα and direct delivery of TGFα‐containing vesicles to the basolateral surface of polarized epithelial cells. VIP and PGE₂ rapidly activate protein kinase A (PKA) that then phosphorylates NKD2 at Ser‐223, a process that is facilitated by the molecular scaffold A‐kinase anchoring protein 12 (AKAP12). This phosphorylation stabilized NKD2, ensuring efficient cell‐surface delivery of TGFα and increased EGFR activation. Thus, GPCR‐triggered, PKA/AKAP12/NKD2‐regulated targeting of TGFα to the cell surface represents a new mode of EGFR transactivation that occurs proximal to ligand cleavage by MMPs.      

Interferon-dependent activation of the serine kinase PI 3'-kinase requires engagement of the IRS pathway but not the Stat pathway

Several signaling pathways are activated by interferon alpha (IFNalpha) in hematopoietic cells, including the Jak-Stat and the insulin receptor substrate (IRS) pathways. It has been previously shown that IFNalpha activates the phosphatidylinositol (PI) 3'-kinase via an interaction of the p85 subunit of PI 3'-kinase with IRS proteins. Other studies have proposed that Stat-3 also functions as an adapter for p85. We sought to identify the major pathway that regulates IFNalpha activation of the PI3'-kinase in hematopoietic cells. Our data demonstrate that IFNalpha induces the interaction of p85 with IRS-1 or IRS-2, but not Stat-3, in various hematopoietic cell lines in which IRS-1 and/or IRS-2 and Stat-3 are activated by IFNalpha. In addition, inhibition of PI 3'-kinase activity by preincubation of cells with the PI 3'-kinase inhibitor LY294002 does not affect IFN-dependent formation of SIF complexes that contain Stat-3. To determine whether phosphorylation of tyrosine residues in the IFN receptor is required for activation of the PI 3'-kinase, we performed studies using mouse L929 fibroblasts transfected with mutated human IFNAR1 and/or IFNAR2 subunits of the Type I IFN receptor, lacking tyrosine phosphorylation sites. The serine kinase activity of the PI-3K was activated by human IFNalpha in these cells, suggesting that phosphorylation of the Type I IFN receptor is not essential for PI3K activation. We then determined whether IFNalpha activates the Akt kinase, a known downstream target for PI 3'-kinase that mediates anti-apoptotic signals. Akt was activated by insulin or IGF-1, but not IFNalpha, in the IFNalpha-sensitive U-266 myeloma cell line. Altogether, our data establish that the IRS pathway and not the Stat pathway, is the major pathway regulating engagement of PI 3'-kinase in hematopoietic cells. Furthermore, the selective activation of Akt by insulin/IGF-1 suggests the existence of distinct regulatory activities of PI3'-kinase in growth factor versus interferon signaling.     

Insulin-like growth factor-1 (IGF-1) inhibits the basolateral Cl channels in the thick ascending limb of the rat kidney

The aim of the present study is to test the hypothesis that insulin-like-growth factor-1 (IGF-1) plays a role in the regulation of basolateral Cl channels in the thick ascending limb (TAL). The patch-clamp experiments demonstrated that application of IGF-I or insulin inhibited the basolateral 10-pS Cl channels. However, the concentration of insulin required for the inhibition of the Cl channels by 50% (K(1/2)) was ten times higher than those of IGF-1. The inhibitory effect of IGF-I on the 10-pS Cl channels was blocked by suppressing protein tyrosine kinase or by blocking phosphoinositide 3-kinase (PI3K). In contrast, inhibition of phospholipase C (PLC) failed to abolish the inhibitory effect of IGF-1 on the Cl channels in the TAL. Western blot analysis demonstrated that IGF-1 significantly increased the phosphorylation of phospholipid-dependent kinase (PDK) at serine residue 241 (Ser(241)) and AKT at Ser(473) in the isolated medullary TAL. Moreover, inhibition of PI3K with LY294002 abolished the effect of IGF-1 on the phosphorylation of PDK and AKT. The notion that the effect of IGF-1 on the 10-pS Cl channels was induced by stimulation of PDK-AKT-mTOR pathway was further suggested by the finding that rapamycin completely abolished the effect of IGF-1 on the 10-pS Cl channels in the TAL. We conclude that IGF-1 inhibits the basolateral Cl channels by activating PI3K-AKT-mTOR pathways. The inhibitory effect of IGF-1 on the Cl channels may play a role in ameliorating the ischemia-induced renal injury through IGF-1 administration.     

Comparative signaling pathways of insulin-like growth factor-1 and brain-derived neurotrophic factor in hippocampal neurons and the role of the PI3 kinase pathway in cell survival

Insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor (BDNF) are trophic factors required for the viability and normal functions of various neuronal cells. However, the detailed intracellular mechanism(s) involved in these effects in neuronal cells remains to be fully elucidated. In present study, the respective intracellular signaling pathway induced by IGF-1 and BDNF and their possible role in neuronal survival were investigated. Both IGF-1 and BDNF protected hippocampal neurons from serum deprivation-induced death with IGF-1 apparently being more potent. Western blot analyses showed that both IGF-1 and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3)/Akt (protein kinase B) kinase and the mitogen-activated protein kinase (MAPK) pathways. The phosphorylation of Akt and its downstream target, FKHRL1, induced by IGF-1 was rapid and sustained while that of MAPK was transient. The reverse situation was observed for BDNF. Moreover, IGF-1 potently induced the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with PI3 kinase while BDNF was weak in these assays. In contrast, the tyrosine phosphorylation of Shc proteins was dramatically stimulated by BDNF, with IGF-1 having only a minimal effect. Most interestingly, only the inhibitor of the PI3K/Akt pathway, LY294002, was able to block the survival effects of both IGF-1 and BDNF; an inhibitor of the MAPK pathway inhibitor, PD98059, being ineffective. Taken together, these data reveal that the survival properties of both IGF-1 and BDNF against serum deprivation are mediated by the activation of the PI3K/Akt, but not the MAPK, pathway in hippocampal neurons.     

Activation of the Insulin-like Growth Factor Type 1 Receptor by Deletion of Amino Acids 870–905

We have created a deletion mutant of the insulin-like growth factor type 1 receptor (IGF-1 R) which lacks the 36 amino acids (aa) immediately N-terminal to the transmembrane domain (Δ870–905 IGF-1 R). This region has been reported to have a negative effect on the transforming potential of an avian sarcoma virus gag-IGF-1 R fusion protein. We have sought to determine whether this region plays a similar role in the intact IGF-1 R. Analysis of the tyrosine kinase activity of the Δ870–905 IGF-1 R shows that the mutant receptor is autophosphorylated without IGF-1 stimulation, indicating that the tyrosine kinase domain is constitutively active. In addition, processing of the receptor is decreased, resulting in accumulation of a high molecular weight proreceptor containing both α and β-subunits. A well-characterized substrate of the IGF-1 R, IRS-1, is constitutively phosphorylated by the Δ870–905 IGF-1 R and phosphoinositide (PI) 3-kinase activity, which is normally activated by the phosphorylation of IRS-1 following IGF-1 stimulation, is increased even in the absence of IGF-1. A second intracellular signal pathway normally activated by IGF-1, the MAP kinase pathway, showed no increase in activity in the absence of IGF-1. The Δ870–905 IGF-1 R promoted cell proliferation only in the presence of IGF-1. We conclude that this deletion increases the basal activity of the IGF-1 receptor tyrosine kinase and activates PI 3-kinase, but is unable to stimulate MAP kinase in the absence of ligand. These results confirm those seen in the gag-IGF-1 R fusion protein and indicate that aa 870–905 exert a negative effect on the tyrosine kinase domain of the β-subunit of the IGF-1 R.     

Enhancement of insulin-induced PI3K/Akt/GSK-3beta and ERK signaling by neuronal nicotinic receptor/PKC-alpha/ERK pathway: up-regulation of IRS-1/-2 mRNA and protein in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells treated with nicotine (10 µm for 24 h), phosphorylation of Akt, glycogen synthase kinase‐3β (GSK‐3β) and extracellular signal‐regulated kinase (ERK)1/2 induced by insulin (100 nm for 10 min) was enhanced by ～ 62%, without altering levels of these protein kinases. Nicotine produced time (> 12 h)‐ and concentration (EC₅₀ 3.6 and 13 µm )‐dependent increases in insulin receptor substrate (IRS)‐1 and IRS‐2 levels by ～ 125 and 105%, without altering cell surface density of insulin receptors. In these cells, insulin‐induced tyrosine phosphorylation of IRS‐1/IRS‐2 and recruitment of phosphoinositide 3‐kinase (PI3K) to IRS‐1/IRS‐2 were augmented by ～ 63%. The increase in IRS‐1/IRS‐2 levels induced by nicotine was prevented by nicotinic acetylcholine receptor (nAChR) antagonists, the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetra‐acetic acid tetrakis‐acetoxymethyl ester, cycloheximide or actinomycin D. Nicotine increased IRS‐1 and IRS‐2 mRNA levels by ～ 57 and ～ 50%, and this was prevented by conventional protein kinase C (cPKC) inhibitor Gö6976, or ERK kinase inhibitors PD98059 and U0126. Nicotine phosphorylated cPKC‐α, thereby increasing phosphorylation of ERK1/ERK2, as demonstrated by using Gö6976, PD98059 or U0126. Selective activation of cPKC‐α by thymeleatoxin mimicked these effects of nicotine. Thus, stimulation of nAChRs up‐regulated expression of IRS‐1/IRS‐2 via Ca²⁺‐dependent sequential activation of cPKC‐α and ERK, and enhanced insulin‐induced PI3K/Akt/GSK‐3β and ERK signaling pathways.     

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.     

A novel functional crosstalk between DDR1 and the IGF axis and its relevance for breast cancer

ABSTRACT

In the last decades increasing importance has been attributed to the Insulin/Insulin-like Growth Factor signaling (IIGFs) in cancer development, progression and resistance to therapy. In fact, IIGFs is often deregulated in cancer. In particular, the mitogenic insulin receptor isoform A (IR-A) and the insulin-like growth factor receptor (IGF-1R) are frequently overexpressed in cancer together with their cognate ligands IGF-1 and IGF-2. Recently, we identified discoidin domain receptor 1 (DDR1) as a new IR-A interacting protein. DDR1, a non-integrin collagen tyrosine kinase receptor, is overexpressed in several malignancies and plays a role in cancer progression and metastasis.

Herein, we review recent findings indicating that DDR1 is as a novel modulator of IR and IGF-1R expression and function. DDR1 functionally interacts with IR and IGF-1R and enhances the biological actions of insulin, IGF-1 and IGF-2. Conversely, DDR1 is upregulated by IGF-1, IGF-2 and insulin through the PI3K/AKT/miR-199a-5p circuit. Furthermore, we discuss the role of the non-canonical estrogen receptor GPER1 in the DDR1-IIGFs crosstalk. These data suggest a wider role of DDR1 as a regulator of cell response to hormones, growth factors, and signals coming from the extracellular matrix.