Transactivation of ErbB Family of Receptor Tyrosine Kinases Is Inhibited by Angiotensin-(1-7) via Its Mas Receptor

Transactivation of the epidermal growth factor receptor (EGFR or ErbB) family members, namely EGFR and ErbB2, appears important in the development of diabetes-induced vascular dysfunction. Angiotensin-(1–7) [Ang-(1–7)] can prevent the development of hyperglycemia-induced vascular complications partly through inhibiting EGFR transactivation. Here, we investigated whether Ang-(1–7) can inhibit transactivation of ErbB2 as well as other ErbB receptors in vivo and in vitro. Streptozotocin-induced diabetic rats were chronically treated with Ang-(1–7) or AG825, a selective ErbB2 inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in cultured vascular smooth muscle cells (VSMCs). Ang-(1–7) or AG825 treatment inhibited diabetes-induced phosphorylation of ErbB2 receptor at tyrosine residues Y1221/22, Y1248, Y877, as well as downstream signaling via ERK1/2, p38 MAPK, ROCK, eNOS and IkB-α in the mesenteric vascular bed. In VSMCs cultured in high glucose (25 mM), Ang-(1–7) inhibited src-dependent ErbB2 transactivation that was opposed by the selective Mas receptor antagonist, D-Pro⁷-Ang-(1–7). Ang-(1–7) via Mas receptor also inhibited both Angiotensin II- and noradrenaline/norephinephrine-induced transactivation of ErbB2 and/or EGFR receptors. Further, hyperglycemia-induced transactivation of ErbB3 and ErbB4 receptors could be attenuated by Ang-(1–7) that could be prevented by D-Pro⁷-Ang-(1–7) in VSMC. These data suggest that Ang-(1–7) via its Mas receptor acts as a pan-ErbB inhibitor and might represent a novel general mechanism by which Ang-(1–7) exerts its beneficial effects in many disease states including diabetes-induced vascular complications.     

Regulation and Identification of Na,K-ATPase α1 Subunit Phosphorylation in Rat Parotid Acinar Cells

The stimulation of fluid and electrolyte secretion in salivary cells results in ionic changes that promote rapid increases in the activity of the Na,K-ATPase. In many cell systems, there are conflicting findings concerning the regulation of the phosphorylation of the Na,K-ATPase α subunit, which is the catalytic moiety. Initially, we investigated the phosphorylation sites on the α1 subunit in native rat parotid acinar cells using tandem mass spectrometry and identified two new phosphorylation sites (Ser²²², Ser⁴⁰⁷), three sites (Ser²¹⁷, Tyr²⁶⁰, Ser⁴⁷) previously found from large scale proteomic screens, and two sites (Ser²³, Ser¹⁶) known to be phosphorylated by PKC. Subsequently, we used phospho-specific antibodies to examine the regulation of phosphorylation on Ser²³ and Ser¹⁶ and measured changes in ERK phosphorylation in parallel. The G-protein-coupled muscarinic receptor mimetic carbachol, the phorbol ester phorbol 12-myristate 13-acetate, the Ca²⁺ ionophore ionomycin, and the serine/threonine phosphatase inhibitor calyculin A increased Ser²³ α1 phosphorylation. Inhibition of classical PKC proteins blocked carbachol-stimulated Ser²³ α1 subunit phosphorylation but not ERK phosphorylation, which was blocked by an inhibitor of novel PKC proteins. The carbachol-initiated phosphorylation of Ser²³ α1 subunit was not modified by ERK or PKA activity. The Na,K-ATPase inhibitor ouabain reduced and enhanced the carbachol-promoted phosphorylation of Ser²³ and Ser¹⁶, respectively, the latter because ouabain itself increased Ser¹⁶ phosphorylation; thus, both sites display conformational-dependent phosphorylation changes. Ouabain-initiated phosphorylation of Ser¹⁶ α1 was not blocked by PKC inhibitors, unlike carbachol- or phorbol 12-myristate 13-acetate-initiated phosphorylations, suggesting that this site was also a substrate for a kinase other than PKC.     

Glycogen Synthase Kinase-3β Stabilizes the Interleukin (IL)-22 Receptor from Proteasomal Degradation in Murine Lung Epithelia

Signaling through the interleukin (IL)-22 cytokine axis provides essential immune protection in the setting of extracellular infection as part of type 17 immunity. Molecular regulation of IL-22 receptor (IL-22R) protein levels is unknown. In murine lung epithelia, IL-22R is a relatively short-lived protein (t_½ ∼1.5 h) degraded by the ubiquitin proteasome under normal unstimulated conditions, but its degradation is accelerated by IL-22 treatment. Lys⁴⁴⁹ within the intracellular C-terminal domain of the IL-22R serves as a ubiquitin acceptor site as disruption of this site by deletion or site-directed mutagenesis creates an IL-22R variant that, when expressed in cells, is degradation-resistant and not ubiquitinated. Glycogen synthase kinase (GSK)-3β phosphorylates the IL-22R within a consensus phosphorylation signature at Ser⁴¹⁰ and Ser⁴¹⁴, and IL-22 treatment of cells triggers GSK-3β inactivation. GSK-3β overexpression results in accumulation of IL-22R protein, whereas GSK-3β depletion in cells reduces levels of the receptor. Mutagenesis of IL-22R at Ser⁴¹⁰ and Ser⁴¹⁴ results in receptor variants that display reduced phosphorylation levels and are more labile as compared with wild-type IL-22R when expressed in cells. Further, the cytoskeletal protein cortactin, which is important for epithelial spreading and barrier formation, is phosphorylated and activated at the epithelial cell leading edge after treatment with IL-22, but this effect is reduced after GSK-3β knockdown. These findings reveal the ability of GSK-3β to modulate IL-22R protein stability that might have significant implications for cytoprotective functions and therapeutic targeting of the IL-22 signaling axis.     

Cross-talk between Rho-associated Kinase and Cyclic Nucleotide-dependent Kinase Signaling Pathways in the Regulation of Smooth Muscle Myosin Light Chain Phosphatase

Ca²⁺ sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr⁶⁹⁷ and/or Thr⁸⁵⁵ (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser⁶⁹⁶ prevents phosphorylation at Thr⁶⁹⁷. However, the effects of Ser⁸⁵⁴ and dual Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser⁶⁹⁶, Thr⁶⁹⁷, Ser⁸⁵⁴, and Thr⁸⁵⁵), Ser phosphorylation events (Ser⁶⁹⁶/Ser⁸⁵⁴) and dual Ser/Thr phosphorylation events (Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵). Dual phosphorylation at Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr⁶⁹⁷ and Thr⁸⁵⁵ by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser⁶⁹⁶, Thr⁶⁹⁷, Ser⁸⁵⁴, and Thr⁸⁵⁵ in rat caudal artery, whereas U46619 induced Thr⁶⁹⁷ and Thr⁸⁵⁵ phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.     

PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser^485/491 of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser^485/491 phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser^485/491 phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser^485/491 phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser⁴⁹¹ in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser⁴⁹¹ that plays a negative role in insulin signaling in muscle cells.     

Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase C? Regulates Ras/Mitogen-activated Protein Kinase Signaling and Neuronal Differentiation

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser⁷⁷⁹ in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser⁷⁷⁹ in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser⁷⁷⁹ was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCϵ can phosphorylate Ser⁷⁷⁹ in vitro, whereas overexpression of PKCϵ results in constitutive Ser⁷⁷⁹ phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCϵ reduces both growth factor-induced Ser⁷⁷⁹ phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser⁷⁷⁹, can quantitatively control Ras/MAPK signaling to promote specific cellular responses.     

Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with Toxoplasma gondii

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked IFN-γ. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, 5 µM) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine (5 µM) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, 20 µM) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, 10 µM) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.     

The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser359 Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity

The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr³¹³-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKCδ''s Tyr³¹³-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKCδ''s enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser³⁵⁹. We show that wild-type (WT) PKCδ displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser^359. In contrast, PKCδ-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser³⁵⁹ phosphorylation on native PKCδ and that PKCδ-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr³¹³ docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity.     

A Key Role for the Phosphorylation of Ser440 by the Cyclic AMP-dependent Protein Kinase in Regulating the Activity of the Src Homology 2 Domain-containing Inositol 5′-Phosphatase (SHIP1)

The Src homology 2 domain-containing inositol 5′-phosphatase 1 (SHIP1) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate to phophatidylinositol 3,4-bisphosphate in hematopoietic cells to regulate multiple cell signaling pathways. SHIP1 can be phosphorylated by the cyclic AMP-dependent protein kinase (PKA), resulting in an increase in SHIP1 activity (Zhang, J., Walk, S. F., Ravichandran, K. S., and Garrison, J. C. (2009) J. Biol. Chem. 284, 20070–20078). Using a combination of approaches, we identified the serine residue regulating SHIP1 activity. After mass spectrometric identification of 17 serine and threonine residues on SHIP1 as being phosphorylated by PKA in vitro, studies with truncation mutants of SHIP1 narrowed the phosphorylation site to the catalytic region between residues 400 and 866. Of the two candidate phosphorylation sites located in this region (Ser⁴⁴⁰ and Ser⁷⁷⁴), only mutation of Ser⁴⁴⁰ to Ala abolished the ability of PKA to phosphorylate the purified, catalytic domain of SHIP1 (residues 401–866). Mutation of Ser⁴⁴⁰ to Ala in full-length SHIP1 abrogated the ability of PKA to increase the activity of SHIP1 in mammalian cells. Using flow cytometry, we found that the PKA activator, Sp-adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt hydrate (Sp-cAMPS) blunted the phosphorylation of Akt downstream of B cell antigen receptor engagement in SHIP1-null DT40 B lymphocytes expressing native mouse SHIP1. The inhibitory effect of Sp-cAMPS was absent in cells expressing the S440A mutant of SHIP1. These results suggest that activation of SHIP1 by PKA via phosphorylation on Ser⁴⁴⁰ is an important regulatory event in hematopoietic cells.     

Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue

The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1–3 kinases are specifically activated by two phosphorylation events on residues Thr³⁰⁸ and Ser⁴⁷³ upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser⁴⁷³ and Thr³⁰⁸ phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser⁴⁷³ and Thr³⁰⁸ phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser⁴⁷³-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons.     

Coarse-Grained Molecular Simulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Multi-Site Self-Phosphorylation

Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR (HER/ErbB) family receptors and growth factor receptor PTKs in general.     

TNFα Signals via p66Shc to Induce E-Selectin,Promote Leukocyte Transmigration and Enhance Permeability in Human Endothelial Cells

Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66^Shc acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66^Shc in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66^Shc on Ser³⁶ and the stress kinase c-Jun NH₂-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66^Shc in HUVEC resulted in enhanced p66^Shc phosphorylation on Ser³⁶, increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66^Shc protein, in which Ser³⁶ was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66^Shc resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66^Shc acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66^Shc on Ser³⁶.     

Cyclic-GMP-Dependent Protein Kinase Inhibits the Ras/Mitogen-Activated Protein Kinase Pathway

Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphate and 8-bromoguanosine-3′,5′-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Iβ expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser⁴³, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser⁴³ uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser⁴³ was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser⁴³ in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser⁴³ phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by phosphorylating c-Raf kinase on Ser⁴³ and thereby inhibiting its activation and (ii) by inducing MAP kinase phosphatase 1 expression.     

Identification of Multiple Phosphorylation Sites on Maize Endosperm Starch Branching Enzyme IIb,a Key Enzyme in Amylopectin Biosynthesis

Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser⁶⁴⁹, Ser²⁸⁶, and Ser²⁹⁷. Two Ca²⁺-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser⁶⁴⁹ and Ser²⁸⁶ phosphorylation, and K2, responsible for Ser⁶⁴⁹ and Ser²⁹⁷ phosphorylation. The Ser²⁸⁶ and Ser²⁹⁷ phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel β-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser²⁹⁷ forms a stable salt bridge with Arg⁶⁶⁵, part of a conserved Cys-containing domain in plant branching enzymes. Ser⁶⁴⁹ conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application.     

ErbB2 and ErbB4 Cbl binding sites can functionally replace the ErbB1 Cbl binding site

Poor downregulation of ErbB receptors is associated with enhanced downstream signaling and tumorigenesis. It has been suggested that poor downregulation of ErbB-2, -3 and -4 receptors when compared to ErbB1 is due to decreased recruitment of Cbl E3 ligase proteins. However, a highly conserved Cbl binding site is not only present in ErbB1/EGFR (FLQRpY¹⁰⁴⁵SSDP), but also in ErbB2 (PLQRpY¹⁰⁹¹SEDP) and ErbB4 (STQRpY¹¹⁰³SADP). We therefore replaced the ErbB1 Cbl binding site by that of ErbB2 and ErbB4. Whereas retrovirally infected NIH3T3 cells containing the EGFR Y1045F mutation showed dramatically impaired Cbl recruitment, EGFR ubiquitination and delayed EGFR degradation, replacement of the EGFR Cbl binding site by that of ErbB2 or ErbB4 did not affect Cbl recruitment, receptor-ubiquitination, -degradation, -downregulation or ligand degradation. We conclude that poor downregulation of ErbB2 and ErbB4 receptors is not due to sequence variations in the Cbl binding site of these receptors.     

Gangliosides influence EGFR/ErbB2 heterodimer stability but they do not modify EGF-dependent ErbB2 phosphorylation

Gangliosides are well-known regulators of cell differentiation through specific interactions with growth factor receptors. Previously, our group provided the first evidence about stable association of ganglioside GM₃ to EGFR/ErbB2 heterodimers in mammary epithelial cells. Goals of the present study were to better define the role of gangliosides in EGFR/ErbB2 heterodimerization and receptor phosphorylation events and to analyze their involvement in mammary cell differentiation. Experiments have been conducted using the ceramide analogue (+/−)-treo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ceramide glucosyltransferase resulting in the endogenous ganglioside depletion, and the lactogenic hormone mix DIP (dexamethasone, insulin, prolactin), which induces cell differentiation and β-casein mRNA synthesis. In addition, treatments of ganglioside-depleted cells with exogenous GM₃ have been carried out to ascertain the specific involvement of this ganglioside. Results from co-immunoprecipitation and Western blot experiments have shown that the endogenous ganglioside depletion resulted in the disappearance of SDS-stable EGFR/ErbB2 heterodimers and in the appearance of tyrosine-phosphorylated EGFR also in the absence of EGF stimulation; exogenous GM₃ added in combination with [D]-PDMP reversed both these effects. In contrast, the tyrosine phosphorylation of ErbB2 in ganglioside-depleted cells occurred only after EGF stimulation. Moreover, when ganglioside-depleted cells were treated with DIP in absence of EGF, β-casein gene expression appeared strongly down-regulated, and β-casein mRNA levels were partially restored by exogenous GM₃ treatment. Altogether, although the involvement of other ganglioside species cannot be excluded, these findings sustain the ganglioside GM₃ as an essential molecule for EGFR/ErbB2 heterodimer stability and important regulator of EGFR tyrosine phosphorylation, but it is not crucial for tyrosine phosphorylation of the heterodimerization partner ErbB2. Moreover, modulation of EGFR phosphorylation may explain how gangliosides contribute to regulate the lactogenic hormone-induced mammary cell differentiation.     

Direct Binding of the EGF-like Domain of Neuregulin-1 to Integrins (αvβ3 and α6β4) Is Involved in Neuregulin-1/ErbB Signaling

Integrin-growth factor receptor cross-talk plays a role in growth factor signaling, but the specifics are unclear. In a current model, integrins and growth factor receptors independently bind to their ligands (extracellular matrix and growth factors, respectively). We discovered that neuregulin-1 (NRG1), either as an isolated EGF-like domain or as a native multi-domain form, binds to integrins αvβ3 (with a K_D of 1.36 × 10⁻⁷ m) and α6β4. Docking simulation predicted that three Lys residues at positions 180, 184, and 186 of the EGF-like domain are involved in integrin binding. Mutating these residues to Glu individually or in combination markedly suppressed integrin binding and ErbB3 phosphorylation. Mutating all three Lys residues to Glu (the 3KE mutation) did not affect the ability of NRG1 to bind to ErbB3 but markedly reduced the ability of NRG1 to induce ErbB3 phosphorylation and AKT and Erk1/2 activation in MCF-7 and T47D human breast cancer cells. This suggests that direct integrin binding to NRG1 is critical for NRG1/ErbB signaling. Notably, stimulation of cells with WT NRG1 induced co-precipitation of ErbB3 with α6β4 and with αvβ3 to a much lower extent. This suggests that WT NRG1 induces integrin-NRG1-ErbB3 ternary complex formation. In contrast, the 3KE mutant was much less effective in inducing ternary complex formation than WT NRG1, suggesting that this process depends on the ability of NRG1 to bind to integrins. These results suggest that direct NRG1-integrin interaction mediates integrin-ErbB cross-talk and that α6β4 plays a major role in NRG-ErbB signaling in these cancer cells.     

Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

Diabetes mellitus leads to vascular complications but the underlying signalling mechanisms are not fully understood. Here, we examined the role of ErbB2 (HER2/Neu), a transmembrane receptor tyrosine kinase of the ErbB/EGFR (epidermal growth factor receptor) family, in mediating diabetes-induced vascular dysfunction in an experimental model of type 1 diabetes. Chronic treatment of streptozotocin-induced diabetic rats (1 mg/kg/alt diem) or acute, ex-vivo (10⁻⁶, 10⁻⁵ M) administration of AG825, a specific inhibitor of ErbB2, significantly corrected the diabetes-induced hyper-reactivity of the perfused mesenteric vascular bed (MVB) to the vasoconstrictor, norephinephrine (NE) and the attenuated responsiveness to the vasodilator, carbachol. Diabetes led to enhanced phosphorylation of ErbB2 at multiple tyrosine (Y) residues (Y1221/1222, Y1248 and Y877) in the MVB that could be attenuated by chronic AG825 treatment. Diabetes- or high glucose-mediated upregulation of ErbB2 phosphorylation was coupled with activation of Rho kinases (ROCKs) and ERK1/2 in MVB and in cultured vascular smooth muscle cells (VSMC) that were attenuated upon treatment with either chronic or acute AG825 or with anti-ErbB2 siRNA. ErbB2 likley heterodimerizes with EGFR, as evidenced by increased co-association in diabetic MVB, and further supported by our finding that ERK1/2 and ROCKs are common downstream effectors since their activation could also be blocked by AG1478. Our results show for the first time that ErbB2 is an upstream effector of ROCKs and ERK1/2 in mediating diabetes-induced vascular dysfunction. Thus, potential strategies aimed at modifying actions of signal transduction pathways involving ErbB2 pathway may prove to be beneficial in treatment of diabetes-induced vascular complications.