Impact of Ca2+ on structure of soybean CDPKβ and accessibility of the Tyr-24 autophosphorylation site

Several plant CDPKs were recently shown to be dual specificity kinases rather than Ser/Thr kinases as traditionally classified by sequence analysis. In the present study we confirm the autophosphorylation of recombinant soybean His₆-GmCDPKβ at the Tyr-24 site using sequence- and modification- specific antibodies. Homology modeling of soybean CDPKβ based on recent structures determined for several apicomplexan CDPKs suggested that phosphotyrosine-24 may be inaccessible to phosphatases. However, we report that dephosphorylation of CDPKβ by the protein tyrosine phosphatase 1B, PTP1B, was not restricted in the presence of calcium. Thus, despite conformational changes likely associated with calcium binding to the CDPKs, phosphotyrosine sites remain fully accessible to dephosphorylation suggesting the possibility of conformational breathing and flexing.     

A Conserved Rod Domain Phosphotyrosine That Is Targeted by the Phosphatase PTP1B Promotes Keratin 8 Protein Insolubility and Filament Organization

Post-translational modifications are important functional determinants for intermediate filament (IF) proteins. Phosphorylation of IF proteins regulates filament organization, solubility, and cell-protective functions. Most known IF protein phosphorylation sites are serines localized in the variable “head” and “tail” domain regions. By contrast, little is known about site-specific tyrosine phosphorylation or its implications on IF protein function. We used available proteomic data from large scale studies to narrow down potential phospho-tyrosine sites on the simple epithelial IF protein keratin 8 (K8). Validation of the predicted sites using a pan-phosphotyrosine and a site-specific antibody, which we generated, revealed that the highly conserved Tyr-267 in the K8 “rod” domain was basally phosphorylated. The charge at this site was critically important, as demonstrated by altered filament organization of site-directed mutants, Y267F and Y267D, the latter exhibiting significantly diminished solubility. Pharmacological inhibition of the protein-tyrosine phosphatase PTP1B increased K8 Tyr-267 phosphorylation, decreased solubility, and increased K8 filament bundling, whereas PTP1B overexpression had the opposite effects. Furthermore, there was significant co-localization between K8 and a “substrate-trapping” mutant of PTP1B (D181A). Because K8 Tyr-267 is conserved in many IFs (QYE motif), we tested the effect of the paralogous Tyr in glial fibrillary acidic protein (GFAP), which is mutated in Alexander disease (Y242D). Similar to K8, Y242D GFAP exhibited highly irregular filament organization and diminished solubility. Our results implicate the rod domain QYE motif tyrosine as an important determinant of IF assembly and solubility properties that can be dynamically modulated by phosphorylation.     

Protein-tyrosine Phosphatase 1B Antagonized Signaling by Insulin-like Growth Factor-1 Receptor and Kinase BRK/PTK6 in Ovarian Cancer Cells

Ovarian cancer, which is the leading cause of death from gynecological malignancies, is a heterogeneous disease known to be associated with disruption of multiple signaling pathways. Nevertheless, little is known regarding the role of protein phosphatases in the signaling events that underlie the disease; such knowledge will be essential to gain a complete understanding of the etiology of the disease and how to treat it. We have demonstrated that protein-tyrosine phosphatase 1B (PTP1B) was underexpressed in a panel of ovarian carcinoma-derived cell lines, compared with immortalized human ovarian surface epithelial cell lines. Stable restoration of PTP1B in those cancer cell lines substantially decreased cell migration and invasion, as well as proliferation and anchorage-independent survival. Mechanistically, the pro-survival IGF-1R signaling pathway was attenuated upon ectopic expression of PTP1B. This was due to dephosphorylation by PTP1B of IGF-1R β-subunit and BRK/PTK6, an SRC-like protein-tyrosine kinase that physically and functionally interacts with the IGF-1R β-subunit. Restoration of PTP1B expression led to enhanced activation of BAD, one of the major pro-death members of the BCL-2 family, which triggered cell death through apoptosis. Conversely, inhibition of PTP1B with a small molecular inhibitor, MSI-1436, increased proliferation and migration of immortalized HOSE cell lines. These data reveal an important role for PTP1B as a negative regulator of BRK and IGF-1Rβ signaling in ovarian cancer cells.     

Protein phosphorylation in rat cardiac microsomes: Effects of inhibitors of protein kinase A and of phosphatases

The phosphorylation of rat cardiac microsomal proteins was investigated with special attention to the effects of okadaic acid (an inhibitor of protein phosphatases), inhibitor 2 of protein phosphatase 1 and inhibitor of cyclic AMP-dependent protein kinase (protein kinase A). The results showed that okadaic acid (5 µM) modestly but reproducibly augmented the protein kinase A-catalyzed phospholamban (PLN) phosphorylation, although exerted little effect on the calcium/calmodulin kinase-catalyzed PLN phosphorylation. Microsomes contained three other substrates (M_r 23, 19 and 17 kDa) that were phosphorylated by protein kinase A but not by calcium/calmodulin kinase. The protein kinase A-catalyzed phosphorylation of these three substrates was markedly (2-3 fold) increased by 5 µM okadaic acid. Calmodulin was found to antagonize the action of okadaic acid on such phosphorylation. Protein kinase A inhibitor was found to decrease the protein kinase A-catalyzed phosphorylation of microsomal polyp eptides. Unexpectedly, inhibitor 2 was also found to markedly decrease protein kinase A-catalyzed phosphorylation of phospholamban as well these other microsomal substrates. These results are consistent with the views that protein phosphatase 1 is capable of dephosphorylating membrane-associated phospholamban when it is phosphorylated by protein kinase A, but not by calcium/calmodulin kinase, and that under certain conditions, calcium/calmodulin-stimulated protein phosphatase (protein phosphatase 2B) is also able to dephosphorylate PLN phosphorylated by protein kinase A. Additionally, the observations show that protein phosphatase 1 is extremely active against the three protein kinase A substrates (M_r 23, 19 and 17 kDa) that were present in the isolated microsomes and whose state of phosphorylation was particularly affected in the presence of dimethylsulfoxide. Protein phosphatase 2B is also capable of dephosphorylating these three substrates. (Mol Cell Biochem 175: 109–115, 1997     

Serine phosphorylation of protein tyrosine phosphatase (PTP1B) in HeLa cells in response to analogues of cAMP or diacylglycerol plus okadaic acid

The major intracellular protein tyrosine phosphatase (PTP1B) is a 50kDa protein, localized to the endoplasmic reticulum. This PTP is recovered in the particulate fraction of mamalian cells and can be solubilized as a complex of 150 kDa by extraction with non-ionic detergents. Previous work from this laboratory implicated phosphorylation of serine/threonine residues in the regulation of this PTP. Activity was several-fold higher in cells treated with activators of cAMP-dependent or Ca²⁺/phospholipid-dependent protein kinases or inhibitors of protein phosphatase 2A. Here we show that these treatments result in more than an 8-fold increase in the phosphorylation of the 50kDa PTP catalytic subunit within the 150kDa form of the phosphatase in HeLa cells. The phosphorylation occurred exclusively on serine residues, and the same tryptic and cyanogen bromide,³²P-phosphopeptides were recovered in the PTP from control and stimulated cells. Either multiple kinases phosphorylate a common site in the PTP1B, or a single kinase is activated downstream of cAMP- and Ca²⁺/phospholipid-dependent kinases. The results indicate that phosphorylation of a serine residue in the segment 283–364, probably serine 352 in the sequence Lys-Gly-Ser-Pro-Leu, occurs in response to cell stimulation. Phosphorylation in this region of PTP1B, between the N-terminal catalytic domain and the C-terminal membrane localization segment, is proposed to regulate phosphatase activity.     

Evidence for Reversible Tyrosine Protein Phosphorylation in the Okadaic Acid-Producing Marine Dinoflagellate Prorocentrum lima

ABSTRACT. The protist Prorocentrum lima , a primary producer of the tumour promoter okadaic acid, is a member of the dinoflagellate class of marine microorganisms. Herein, we have identified and characterized a protein tyrosine kinase (designated PLIK 1A) in P. lima that autophosphorylates almost exclusively on tyrosine residues. PLIK 1A was shown to have an approximate molecular mass of 38 kDa by SDS-PAGE and a native molecular mass within the range of 47–55 kDa by Superdex-75 gel filtration. Phosphoamino acid analysis of autophosphorylated PLIK 1A revealed the presence of phosphotyrosine and autophosphorylated PLJK 1A reacted with monoclonal anti-phosphotyrosine antibodies in a Western immunoblot. In addition, two protein tyrosine phosphatases were identified in P. lima that had apparent molecular masses within the ranges of 150–168 kDa and 73–82 kDa as determined by Superdex-200 gel filtration. These P. lima phosphatases, termed PLPTP-I and PLPTP-II, efficiently dephosphorylated tyrosine phosphorylated myelin basic protein. owever, only PLPTP-I was capable of dephosphorylating the tyrosine phosphorylated substrate angiotensin. Both PLPTP-I and PLPTP-II were able to dephosphorylate tyrosine autophosphorylated PLIK 1A. These data provide the first evidence for reversible tyrosine protein phosphorylation in P. lima by protein tyrosine kinases and phosphatases     

A food-derived synergist of NGF signaling: identification of protein tyrosine phosphatase 1B as a key regulator of NGF receptor-initiated signal transduction

Neurotrophins, such as the nerve growth factor (NGF), play an essential role in the growth, development, survival and functional maintenance of neurons in the central and peripheral systems. They also prevent neuronal cell death under various stressful conditions, such as ischemia and neurodegenerative disorders. NGF induces cell differentiation and neurite outgrowth by binding with and activating the NGF receptor tyrosine kinase followed by activation of a variety of signaling cascades. We have investigated the NGF-dependent neuritogenesis enhancer potential of a food-derived small molecule contained in Brassica vegetables and identified the protein tyrosine phosphatase (PTP) 1B as a key regulator of the NGF receptor-initiated signal transduction. Based on an extensive screening of Brassica vegetable extracts for the neuritogenic-promoting activity in the rat pheochromocytoma cell line PC12, we found the Japanese horseradish, wasabi (Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analogue of sulforaphane isolated from broccoli, as one of the major neuritogenic enhancers in the wasabi. 6-HITC strongly enhanced the neurite outgrowth and neurofilament expression elicited by a low-concentration of NGF that alone was insufficient to induce neuronal differentiation. 6-HITC also facilitated the sustained-phosphorylation of the extracellular signal-regulated kinase and the autophosphorylation of the NGF receptor TrkA. It was found that PTP1B act as a phosphatase capable of dephosphorylating Tyr-490 of TrkA and was inactivated by 6-HITC in a redox-dependent manner. The identification of PTP1B as a regulator of NGF signaling may provide new clues about the chemoprotective potential of food components, such as isothiocyanates.     

Phosphorylated alpha-actinin and protein-tyrosine phosphatase 1B coregulate the disassembly of the focal adhesion kinase x Src complex and promote cell migration

The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates a binding site for Src family kinases. FAK phosphorylates the cytoskeletal protein alpha-actinin at Tyr-12. Here we report that protein-tyrosine phosphatase 1B (PTP 1B) is an alpha-actinin phosphatase. PTP 1B-dependent dephosphorylation of alpha-actinin was seen in COS-7 cells and PTP 1B-null fibroblasts reconstituted with PTP 1B. Furthermore, we show that coexpression of wild-type alpha-actinin and PTP 1B causes dephosphorylation at Tyr-397 in FAK. No dephosphorylation was observed in cells coexpressing the alpha-actinin phosphorylation mutant Y12F and PTP 1B. Furthermore, the phosphorylation at four other sites in FAK was not altered by PTP 1B. In addition, we found that phosphorylated alpha-actinin bound to Src and reduced the binding of FAK to Src. The dephosphorylation at Tyr-397 in FAK triggered by wild-type alpha-actinin and PTP 1B caused a significant increase in cell migration. We propose that phosphorylated alpha-actinin disrupts the FAK x Src complex exposing Tyr-397 in FAK to PTP 1B. These findings uncover a novel feedback loop involving phosphorylated alpha-actinin and PTP 1B that regulates FAK x Src interaction and cell migration.     

Long-term high glucose concentration influences Akt, ERK1/2, and PTP1B protein expression in human aortic smooth muscle cells

Hyperglycemia stimulates a plethora of intracellular signaling pathways within the cells of the vascular wall resulting in dysfunction-associated pathologies. Most of the studies reported so far explored the effect of rather short-time exposure of smooth muscle cells to high glucose concentrations. To mimic situation in Type 2 diabetes in which vascular wall is constantly exposed to circulating hyperglycemia, we report here the long-term (7 days) effect of high glucose concentration on human media artery smooth muscle cells. This consists in up-regulation of PTP1B protein expression, down-regulation of basal Akt phosphorylation, and elevation of basal ERK1/2 activation. Acute stimulation of cells in high glucose with insulin down-regulated PTP1B expression, slightly decreased ERK1/2 activity, and activated Akt, whereas oxidative stress up-regulated Akt and ERK1/2 phosphorylation. In conclusion, long-term high glucose and acute oxidative stress and insulin stimulation imbalance the expression of activated kinases Akt and ERK1/2 and of dephosphorylating PTP1B in the insulin signaling pathway.     

UBC9-dependent Association between Calnexin and Protein Tyrosine Phosphatase 1B (PTP1B) at the Endoplasmic Reticulum

Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism.     

Phosphorylation-dependent regulation of T-cell activation by PAG/Cbp,a lipid raft-associated transmembrane adaptor

PAG/Cbp (hereafter named PAG) is a transmembrane adaptor molecule found in lipid rafts. In resting human T cells, PAG is tyrosine phosphorylated and associated with Csk, an inhibitor of Src-related protein tyrosine kinases. These modifications are rapidly lost in response to T-cell receptor (TCR) stimulation. Overexpression of PAG was reported to inhibit TCR-mediated responses in Jurkat T cells. Herein, we have examined the physiological relevance and the mechanism of PAG-mediated inhibition in T cells. Our studies showed that PAG tyrosine phosphorylation and association with Csk are suppressed in response to activation of normal mouse T cells. By expressing wild-type and phosphorylation-defective (dominant-negative) PAG polypeptides in these cells, we found that the inhibitory effect of PAG is dependent on its capacity to be tyrosine phosphorylated and to associate with Csk. PAG-mediated inhibition was accompanied by a repression of proximal TCR signaling and was rescued by expression of a constitutively activated Src-related kinase, implying that it is due to an inactivation of Src kinases by PAG-associated Csk. We also attempted to identify the protein tyrosine phosphatases (PTPs) responsible for dephosphorylating PAG in T cells. Through cell fractionation studies and analyses of genetically modified mice, we established that PTPs such as PEP and SHP-1 are unlikely to be involved in the dephosphorylation of PAG in T cells. However, the transmembrane PTP CD45 seems to play an important role in this process. Taken together, these data provide firm evidence that PAG is a bona fide negative regulator of T-cell activation as a result of its capacity to recruit Csk. They also suggest that the inhibitory function of PAG in T cells is suppressed by CD45. Lastly, they support the idea that dephosphorylation of proteins on tyrosine residues is critical for the initiation of T-cell activation.     

Regulation of the Met receptor-tyrosine kinase by the protein-tyrosine phosphatase 1B and T-cell phosphatase

The non-receptor protein-tyrosine phosphatases (PTPs) 1B and T-cell phosphatase (TCPTP) have been implicated as negative regulators of multiple signaling pathways including receptor-tyrosine kinases. We have identified PTP1B and TCPTP as negative regulators of the hepatocyte growth factor receptor, the Met receptor-tyrosine kinase. In vivo, loss of PTP1B or TCPTP enhances hepatocyte growth factor-mediated phosphorylation of Met. Using substrate trapping mutants of PTP1B or TCPTP, we have demonstrated that both phosphatases interact with Met and that these interactions require phosphorylation of twin tyrosines (Tyr-1234/1235) in the activation loop of the Met kinase domain. Using confocal microscopy, we show that trapping mutants of both PTP1B and the endoplasmic reticulum-targeted TCPTP isoform, TC48, colocalize with Met and that activation of Met enables the nuclear-localized isoform of TCPTP, TC45, to exit the nucleus. Using small interfering RNA against PTP1B and TCPTP, we demonstrate that phosphorylation of Tyr-1234/1235 in the activation loop of the Met receptor is elevated in the absence of either PTP1B or TCPTP and further elevated upon loss of both phosphatases. This enhanced phosphorylation of Met corresponds to enhanced biological activity and cellular invasion. Our data demonstrate that PTP1B and TCPTP play distinct and non-redundant roles in the regulation of the Met receptor-tyrosine kinase.     

Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases

Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC(50) values of 1-8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC(50)=0.1 nM) and MPH (IC(50)=8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways.     

A novel phosphatase family, structurally related to dual-specificity phosphatases, that displays unique amino acid sequence and substrate specificity

Members of the superfamily of protein tyrosine phosphatases (PTPs) share the presence of an evolutionarily conserved PTP catalytic domain. Among them, the dual-specificity phosphatases (DSPs) constitute a diverse group of enzymes in terms of substrate specificity, including nonprotein substrates. In recent years, an increasing number of novel DSPs, whose functions and biological substrates are not well defined, have been discovered in a variety of organisms. In this study, we define the structural and functional properties of evolutionarily related atypical DSPs from different phyla. Sets of conserved motifs were defined that (i) uniquely segregated mammalian atypical DSPs from closely related enzymes and (ii) exclusively characterised a novel family of atypical DSPs present in plants, fungi, and kinetoplastids [plant and fungi atypical (PFA)-DSPs]; despite having different sequence “fingerprints,” the PTP tertiary structure of PFA-DSPs is conserved. Analysis of the catalytic properties of PFA-DSPs suggests the existence of a unique substrate specificity for these enzymes. Our findings predict characteristic functional motifs for the diverse members of the DSP families of PTPs and provide insights into the functional properties of DSPs of unknown function.     

Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B

To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions.     

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.     

PTP inhibitor IV protects JNK kinase activity by inhibiting dual-specificity phosphatase 14 (DUSP14)

Protein phosphorylation plays critical roles in the regulation of protein activity and cell signaling. The level of protein phosphorylation is controlled by protein kinases and protein tyrosine phosphatases (PTPs). Disturbance of the equilibrium between protein kinase and PTP activities results in abnormal protein phosphorylation, which has been linked to the etiology of several diseases, including cancer. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by bis (4-trifluoromethyl-sulfonamidophenyl, TFMS)-1,4-diisopropylbenzene (PTP inhibitor IV). PTP inhibitor IV inhibited DUSP14 phosphatase activity. Kinetic studies with PTP inhibitor IV and DUSP14 revealed a competitive inhibition, suggesting that PTP inhibitor IV binds to the catalytic site of DUSP14. PTP inhibitor IV effectively and specifically inhibited DUSP14-mediated dephosphorylation of JNK, a member of the mitogen-activated protein kinase (MAPK) family.     

Protein tyrosine phosphatase 1B inhibits adipocyte differentiation and mediates TNFα action in obesity

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of systemic glucose and insulin homeostasis; however, its exact role in adipocytes is poorly understood. This study was to elucidate the role of PTP1B in adipocyte differentiation and its implication in obesity. During differentiation of 3T3-L1 white preadipocytes, PTP1B decreased progressively with adipocyte maturation. Lentivirus-mediated PTP1B overexpression in preadipocytes delayed adipocyte differentiation, shown as lack of mature adipocytes, low level of lipid accumulation, and down-regulation of main markers (PPARγ2, SREBP-1c, FAS and LPL). In contrast, lentivirus-mediated PTP1B knockdown accelerated adipocyte differentiation, demonstrated as full of mature adipocytes, high level of lipid accumulation, and up-regulation of main markers. Dominant-negative inhibition on endogenous PTP1B by lentivirus-mediated overexpression of PTP1B double mutant in Tyr-46 and Asp-181 residues (LV-D/A-Y/F) also stimulated adipogenesis, more efficient than PTP1B knockdown. Diet-induced obesity mice exhibited an up-regulation of PTP1B and TNFα accompanied by a down-regulation of PPARγ2 in white adipose tissue. TNFα recombinant protein impeded PTP1B reduction and inhibited adipocyte differentiation in vitro; this inhibitory effect was prevented by LV-D/A-Y/F. Moreover, PTP1B inhibitor treatment improved adipogenesis and suppressed TNFα in adipose tissue of obese mice. All together, PTP1B negatively regulates adipocyte development and may mediate TNFα action to impair adipocyte differentiation in obesity. Our study provides novel evidence for the importance of PTP1B in obesity and for the potential application of PTP1B inhibitors.     

Inhibiting Tyrosine Phosphorylation of Protein Kinase Cδ (PKCδ) Protects the Salivary Gland from Radiation Damage

Radiation therapy for head and neck cancer can result in extensive damage to normal adjacent tissues such as the salivary gland and oral mucosa. We have shown previously that tyrosine phosphorylation at Tyr-64 and Tyr-155 activates PKCδ in response to apoptotic stimuli by facilitating its nuclear import. Here we have identified the tyrosine kinases that mediate activation of PKCδ in apoptotic cells and have explored the use of tyrosine kinase inhibitors for suppression of irradiation-induced apoptosis. We identify the damage-inducible kinase, c-Abl, as the PKCδ Tyr-155 kinase and c-Src as the Tyr-64 kinase. Depletion of c-Abl or c-Src with shRNA decreased irradiation- and etoposide-induced apoptosis, suggesting that inhibitors of these kinases may be useful therapeutically. Pretreatment with dasatinib, a broad spectrum tyrosine kinase inhibitor, blocked phosphorylation of PKCδ at both Tyr-64 and Tyr-155. Expression of “gate-keeper” mutants of c-Abl or c-Src that are active in the presence of dasatinib restored phosphorylation of PKCδ at Tyr-155 and Tyr-64, respectively. Imatinib, a c-Abl-selective inhibitor, also specifically blocked PKCδ Tyr-155 phosphorylation. Dasatinib and imatinib both blocked binding of PKCδ to importin-α and nuclear import, demonstrating that tyrosine kinase inhibitors can inhibit nuclear accumulation of PKCδ. Likewise, pretreatment with dasatinib also suppressed etoposide and radiation induced apoptosis in vitro. In vivo, pre-treatment of mice with dasatinib blocked radiation-induced apoptosis in the salivary gland by >60%. These data suggest that tyrosine kinase inhibitors may be useful prophylactically for protection of nontumor tissues in patients undergoing radiotherapy of the head and neck.