Receptor tyrosine kinases (RTKs) are key regulators of cellular homeostasis. Based on in vitro and ex vivo studies, protein tyrosine phosphatase-1B (PTP1B) was implicated in the regulation of several RTKs, yet mice lacking PTP1B show defects mainly in insulin and leptin receptor signaling. To address this apparent paradox, we studied RTK signaling in primary and immortalized fibroblasts from PTP1B ؊/؊ mice. After growth factor treatment, cells lacking PTP1B exhibit increased and sustained phosphorylation of the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR). However, Erk activation is enhanced only slightly, and there is no increase in Akt activation in PTP1B-deficient cells. Our results show that PTP1B does play a role in regulating EGFR and PDGFR phosphorylation but that other signaling mechanisms can largely compensate for PTP1B deficiency. In-gel phosphatase experiments suggest that other PTPs may help to regulate the EGFR and PDGFR in PTP1Bfibroblasts. This and other compensatory mechanisms prevent widespread, uncontrolled activation of RTKs in the absence of PTP1B and probably explain the relatively mild effects of PTP1B deletion in mice.Regulation of cellular proliferation, adhesion, and migration is pivotal for maintaining homeostasis. Multiple peptide growth factors direct these processes to differing extents in primary fibroblasts and fibroblast cell lines. These growth factors signal via receptors with intrinsic protein-tyrosine kinase activity, termed receptor tyrosine kinases (RTKs).1 Ligand binding activates the intrinsic kinase activity of these receptors, resulting in the phosphorylation of multiple receptor tyrosyl residues. These serve as docking sites to recruit signal relay molecules containing src homology-2 and/or phosphotyrosine binding domains, most of which also are RTK substrates. Based largely on experiments in which wild-type PTPs or their catalytically impaired (dominant-negative) mutants were overexpressed, multiple PTPs, including LAR (7), DEP-1 (8), TC-PTP (9, 10), Shp-1 (11, 12), Shp-2 (13), and PTP1B (14 -17) have been implicated in the dephosphorylation of various RTKs. Which, if any, of these PTPs regulate RTK signaling under physiologically relevant conditions of expression has remained largely unclear. Also unknown is the extent to which RTK dephosphorylation, as opposed to other down-regulatory mechanisms such as RTK degradation and/or inhibitory seryl phosphorylation, plays the (a) key role in receptor inactivation.PTP1B is a widely expressed non-receptor PTP that is localized on intracellular membranes via a hydrophobic C-terminal targeting sequence (18,19). A role for PTP1B in the regulation of many cellular functions has been suggested, including integrin (20 -23), cadherin (24, 25), and cytokine receptor signaling (26 -28), cell cycle regulation (29 -31), and the response to cellular stress (32). Multiple studies indicated that PTP1B dephosphorylates the EGFR (16, 17) and the insulin receptor (IR) (14,(33)(34)(35). Analy...

Constitutive activation of receptor tyrosine kinases (RTKs) is a frequent event in human cancer cells. Activating mutations in Fms-like tyrosine kinase 3 (FLT-3), notably, internal tandem duplications in the juxtamembrane domain (FLT-3 ITD), have been causally linked to acute myeloid leukemia. As we describe here, FLT-3 ITD exists predominantly in an immature, underglycosylated 130-kDa form, whereas wild-type FLT-3 is expressed predominantly as a mature, complex glycosylated 150-kDa molecule. Endogenous FLT-3 ITD, but little wild-type FLT-3, is detectable in the endoplasmic reticulum (ER) compartment. Conversely, cell surface expression of FLT-3 ITD is less efficient than that of wild-type FLT-3. Inhibition of FLT-3 ITD kinase by small molecules, inactivating point mutations, or coexpression with the protein-tyrosine phosphatases (PTPs) SHP-1, PTP1B, and PTP-PEST but not RPTP␣ promotes complex glycosylation and surface localization. However, PTP coexpression has no effect on the maturation of a surface glycoprotein of vesicular stomatitis virus. The maturation of wild-type FLT-3 is impaired by general PTP inhibition or by suppression of endogenous PTP1B. Enhanced complex formation of FLT-3 ITD with the ER-resident chaperone calnexin indicates that its retention in the ER is related to inefficient folding. The regulation of RTK maturation by tyrosine phosphorylation was observed with other RTKs as well, defines a possible role for ER-resident PTPs, and may be related to the altered signaling quality of constitutively active, transforming RTK mutants.Cellular receptors for growth factors, hormones, cytokines, and antigens are postranslationally modified with N-linked, branched carbohydrate chains. Nascent polypeptide chains become initially glycosylated with a mannose-rich branched oligosaccharide in the endoplasmic reticulum (ER). Then, the glycoproteins are subjected to partial deglycosylation by several selective glycosidases, eventually enabling transfer to the Golgi compartment and more complex glycosylation (9). This process, designated glycoprotein maturation, is coupled to stringent quality control in the ER (4, 10). Correct folding is monitored by a complex system comprising, among other components, the chaperones calnexin and calreticulin, the oxidoreductase ERp57, and the glycosylation enzymes UDP-glucose glucosyltransferase and glucosidases I and II. Improperly folded glycoproteins are tagged by reversible glucosylation, enabling their interactions with calnexin and calreticulin and leading to their retention in the ER (4). Properly folded glycoproteins can dissociate from the chaperones and proceed to the Golgi compartment for further glycosylation.The receptor tyrosine kinase (RTK) Fms-like tyrosine kinase 3 (FLT-3) is expressed in multiple hematopoietic lineages (21,22). Constitutively active FLT-3 mutants, notably, versions harboring internal tandem duplications in the juxtamembrane domain (FLT-3 ITD) and versions with point mutations in the kinase activation loop, have been found in approximately ...

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor ␤ (PDGFR␤) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFR␤ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFR␤ in porcine aortic endothelial cells transfected with the ␤-receptor, but not in cells transfected with the ␣-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFR␤. We furthermore visualized tyrosine phosphorylated PDGFR␤ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology. Molecular & Cellular Proteomics 6:1500 -1509, 2007.

The ESAC PPS provided useful information on the quality of prescribing, which identified a number of targets for quality improvement. These could apply to specific departments or whole hospitals. Intensive care, which has different characteristics, should not be compared with general wards with respect to combination therapy, hospital-acquired infections or parenteral proportion. The study confirmed that the ESAC PPS methodology can be used on a large number of hospitals at regional, national, continental or global level.

IntroductionFms-like tyrosine kinase 3 (FLT3) belongs to the class III receptor tyrosine kinase (RTK) family that includes FMS, platelet-derived growth factor receptor (PDGFR), and c-KIT. 1 FLT3 plays an important physiologic role in self-renewal and differentiation of hematopoietic stem and progenitor cells. [2][3][4] In acute myeloid leukemia (AML), activating mutations in the FLT3 gene occur in 30% to 40% of adult patients and have been demonstrated to play a crucial role in driving proliferation and survival of the leukemic clone. Twenty percent to 30% of AML patients harbour an internal tandem duplication (ITD) in the juxtamembrane (JM) region of FLT3. 5,6 ITDs found in FLT3 are always in frame and range from 3 base pairs (bp) to more than 400 bp. 7 Ten percent of AML patients have mutations within the activation loop of the second kinase domain, predominantly substitutions of aspartate at residue 835 (D835). However, additional activating mutations in this region have also been described. [8][9][10] Expression of FLT3_ITD receptors results in autophosphorylation of FLT3 and subsequent activation of downstream signaling. 11-13 Consequently, FLT3_ITD mutations render hematopoietic cells growth factor-independent by promoting cell proliferation and inhibition of apoptosis and lead to myeloproliferative disease in a murine transplantation model. 11,[13][14][15] Generally, it is believed that the ITD insertions within FLT3 occur in the zipper or linker peptide segment of JM (JM-Z) near the JM hinge region, thereby disrupting the autoinhibitory function of the JM-domain. 16 However, a detailed analysis of FLT3_ITD insertion sites has not been performed so far. Therefore, we have addressed this issue and have determined ITD insertion sites of 753 unselected FLT3_ITD-positive AML cases by direct cDNA sequencing. Methods Sequence analysis of FLT3_ITD from 753 unselected FLT3_ITD-positive AML casesMononucleated bone marrow cells from patients participating in standard diagnostic procedures and treated according to protocols of the AML Cooperative Group (AMLCG) study group or according to other intensive AML therapy protocols were obtained by Ficoll density gradient centrifugation. Some of the patients have already been described previously. 7 mRNA was extracted with the MagnaPureLC mRNA Kit I (Roche Diagnostics, Mannheim, Germany). cDNA synthesis of mRNA of an equivalent of 5 to 10 ϫ 10 6 cells was performed using SuperscriptII (Gibco BRL/Invitrogen, Karlsruhe, Germany) and random hexamer primers (Pharmacia, Freiburg, Germany). PCR and evaluation was performed as described previously. 7 Mutation detection was done by standard agarose gel electrophoresis and in parallel by fragment analysis on a capillary sequencer as described previously. 17 Direct sequencing of PCR products was performed as described 7 and primers were the same as used for PCR or RT-PCR amplification, respectively.Blood samples from AML patients were collected after informed consent was obtained in accordance with the Declaration of Helsinki. Labor...

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The platelet-derived growth factor (PDGF) ␤ receptor mediates mitogenic and chemotactic signals. Like other tyrosine kinase receptors, the PDGF ␤ receptor is negatively regulated by protein tyrosine phosphatases (PTPs). To explore whether T-cell PTP (TC-PTP) negatively regulates the PDGF ␤ receptor, we compared PDGF ␤ receptor tyrosine phosphorylation in wild-type and TC-PTP knockout (ko) mouse embryos. PDGF ␤ receptors were hyperphosphorylated in TC-PTP ko embryos. Fivefold-higher ligand-induced receptor phosphorylation was observed in TC-PTP ko mouse embryo fibroblasts (MEFs) as well. Reexpression of TC-PTP partly abolished this difference. As determined with site-specific phosphotyrosine antibodies, the extent of hyperphosphorylation varied among different autophosphorylation sites. The phospholipase C␥1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase C␥1 activity and migratory hyperresponsiveness to PDGF. PDGF ␤ receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTP ko MEFs was also higher than that in control cells. This increase occurred with a site distribution different from that seen after TC-PTP depletion. PDGF-induced migration was not increased in PTP-1B ko cells. In summary, our findings identify TC-PTP as a previously unrecognized negative regulator of PDGF ␤ receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors.Protein tyrosine phosphatases (PTPs) are natural receptor tyrosine kinase antagonists and serve as regulators of both nonreceptor and receptor tyrosine kinases (28,29). Recent investigations indicated that each receptor tyrosine kinase associates with and is dephosphorylated by a number of tyrosine phosphatases. The dephosphorylation of the receptor by individual PTPs can be general, thereby terminating receptor signaling. Alternatively, PTPs can site selectively dephosphorylate a subset of tyrosine residues and thereby modulate signaling downstream of the receptor. By regulating the expression and activation of tyrosine phosphatases, the cell consequently might be able to modulate signaling through receptor tyrosine kinases and fine-tune its response.Platelet-derived growth factors (PDGFs) are a family of growth factors that stimulate cell growth, survival, and motility. PDGF isoforms act by binding to two structurally related protein tyrosine kinases, the PDGF ␣ and ␤ receptors (16). The binding of PDGF to its receptors results in receptor dimerization, promoting phosphorylation in trans between the two receptors in the complex. PDGF-AA forms ␣␣ receptor dimers, PDGF-AB forms ␣␣ and ␣␤ receptor dimers, and PDGF-BB forms all combinations of receptor dimers. Two more PDGF dimers, PDGF-CC and PDGF-DD, recently were identified (2, 24, 25) and shown to preferentially signal through ␣␣ receptor and ␤␤ receptor dimers, respectively, but also may activate both receptor ty...

The aim of this study was to evaluate the prevalence of some virulence genes among 202 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients (n=42) and non-CF in-patients (n=160)