scholarly journals Tyrosine Phosphorylation Regulates Maturation of Receptor Tyrosine Kinases

2005 ◽  
Vol 25 (9) ◽  
pp. 3690-3703 ◽  
Author(s):  
Dirk-E. Schmidt-Arras ◽  
Annette Böhmer ◽  
Boyka Markova ◽  
Chunaram Choudhary ◽  
Hubert Serve ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Human Cancer ◽  
Point Mutations ◽  
Protein Tyrosine Phosphatases ◽  
Surface Glycoprotein ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Frequent Event

ABSTRACT 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 RPTPα 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.

scholarly journals Characterization of an activated mutant of focal adhesion kinase: ‘SuperFAK’

2002 ◽  
Vol 365 (3) ◽  
pp. 591-603 ◽  
Author(s):  
Veronica GABARRA-NIECKO ◽  
Patricia J. KEELY ◽  
Michael D. SCHALLER
Keyword(s):  
Catalytic Activity ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Human Cancer ◽  
Point Mutations ◽  
Enzymic Activity ◽  
Wild Type

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in normal cellular processes such as adhesion, spreading, migration, proliferation and survival. In addition, FAK is overexpressed in a variety of cancer cells and tumours and may play a role in the development of human cancer. As a prelude to modelling the role of aberrant FAK signalling in the initiation of cancer, the goal of the present study was to engineer point mutations in FAK that would enhance enzymic activity. A number of substitutions that were reported as activating mutations in other tyrosine kinases were introduced into FAK. Glutamic acid substitutions for two lysine residues in the activation loop of FAK, based upon the K650E (Lys650→Glu) mutant of fibroblast-growth-factor receptor 3, were made to create ‘SuperFAK'. Two brain-specific exons were engineered into avian FAK to create FAK6.7. SuperFAK and, to a lesser extent, FAK6.7, exhibited increased catalytic activity in vitro compared with wild-type FAK. The expression of SuperFAK and FAK6.7 in fibroblasts led to hyperphosphorylation of FAK substrates. Although the catalytic activity of SuperFAK and FAK6.7 was largely independent of cell adhesion, tyrosine phosphorylation of downstream substrates was adhesion-dependent. Further, since SuperFAK exhibited the same ability as wild-type FAK to recruit Src family kinases, tyrosine phosphorylation of substrates was likely due to direct phosphorylation by FAK. In addition to enhanced biochemical signalling, SuperFAK also increased the motility of epithelial cells. SuperFAK and FAK6.7 may be valuable molecular tools to investigate the potential role of aberrant FAK signalling in human disease.

scholarly journals Noncatalytic Inhibition of Cyclic Nucleotide–gated Channels by Tyrosine Kinase Induced by Genistein

1999 ◽  
Vol 113 (1) ◽  
pp. 45-56 ◽  
Author(s):  
Elena Molokanova ◽  
Alexei Savchenko ◽  
Richard H. Kramer
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cyclic Nucleotide ◽  
Tyrosine Kinases ◽  
Time Course ◽  
Protein Tyrosine Phosphatases ◽  
Competitive Inhibitor ◽  
Rod Photoreceptor ◽  
Protein Tyrosine ◽  
Atp Binding Site ◽  
Cng Channel

Rod photoreceptor cyclic nucleotide–gated (CNG) channels are modulated by tyrosine phosphorylation. Rod CNG channels expressed in Xenopus oocytes are associated with constitutively active protein tyrosine kinases (PTKs) and protein tyrosine phosphatases that decrease and increase, respectively, the apparent affinity of the channels for cGMP. Here, we examine the effects of genistein, a competitive inhibitor of the ATP binding site, on PTKs. Like other PTK inhibitors (lavendustin A and erbstatin), cytoplasmic application of genistein prevents changes in the cGMP sensitivity that are attributable to tyrosine phosphorylation of the CNG channels. However, unlike these other inhibitors, genistein also slows the activation kinetics and reduces the maximal current through CNG channels at saturating cGMP. These effects occur in the absence of ATP, indicating that they do not involve inhibition of a phosphorylation event, but rather involve an allosteric effect of genistein on CNG channel gating. This could result from direct binding of genistein to the channel; however, the time course of inhibition is surprisingly slow (>30 s), raising the possibility that genistein exerts its effects indirectly. In support of this hypothesis, we find that ligands that selectively bind to PTKs without directly binding to the CNG channel can nonetheless decrease the effect of genistein. Thus, ATP and a nonhydrolyzable ATP derivative competitively inhibit the effect of genistein on the channel. Moreover, erbstatin, an inhibitor of PTKs, can noncompetitively inhibit the effect of genistein. Taken together, these results suggest that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel catalyzed by PTKs, genistein triggers a noncatalytic interaction between the PTK and the channel that allosterically inhibits gating.

Activation of pp60(src) is critical for stretch-induced orienting response in fibroblasts

1999 ◽  
Vol 112 (9) ◽  
pp. 1365-1373 ◽  
Author(s):  
X. Sai ◽  
K. Naruse ◽  
M. Sokabe
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Orienting Response ◽  
Cyclic Stretch ◽  
Wild Type ◽  
Herbimycin A ◽  
Molecular Masses

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120–130 kDa) increased and peaked at 30 minutes. Immuno-precipitation experiments revealed that paxillin, pp125(FAK), and pp130(CAS) were included in the 70 kDa, and 120–130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60(src), the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60(src) increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an anti-sense S-oligodeoxynucleotide (S-ODN) against pp60(src), but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60(src), we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1 than in 3Y1 fibroblasts. These results strongly suggest that cyclic stretch induces the activation of pp60(src) and that pp60(src) is indispensable for the tyrosine phosphorylation of pp130(CAS), pp125(FAK) and paxillin followed by the orienting response in 3Y1 fibroblasts.

scholarly journals Activation of Phosphotyrosine-Mediated Signaling Pathways in the Cortex and Spinal Cord of SOD1G93A, a Mouse Model of Familial Amyotrophic Lateral Sclerosis

2018 ◽  
Vol 2018 ◽  
pp. 1-10
Author(s):  
Cinzia Mallozzi ◽  
Alida Spalloni ◽  
Patrizia Longone ◽  
Maria Rosaria Domenici
Keyword(s):  
Spinal Cord ◽  
Amyotrophic Lateral Sclerosis ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Tyrosine Phosphatases ◽  
Direct Stimulation ◽  
Tyrosine Kinase 2 ◽  
Lateral Sclerosis

Degeneration of cortical and spinal motor neurons is the typical feature of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease for which a pathogenetic role for the Cu/Zn superoxide dismutase (SOD1) has been demonstrated. Mice overexpressing a mutated form of the SOD1 gene (SOD1G93A) develop a syndrome that closely resembles the human disease. The SOD1 mutations confer to this enzyme a “gain-of-function,” leading to increased production of reactive oxygen species. Several oxidants induce tyrosine phosphorylation through direct stimulation of kinases and/or phosphatases. In this study, we analyzed the activities of src and fyn tyrosine kinases and of protein tyrosine phosphatases in synaptosomal fractions prepared from the motor cortex and spinal cord of transgenic mice expressing SOD1G93A. We found that (i) protein phosphotyrosine level is increased, (ii) src and fyn activities are upregulated, and (iii) the activity of tyrosine phosphatases, including the striatal-enriched tyrosine phosphatase (STEP), is significantly decreased. Moreover, the NMDA receptor (NMDAR) subunit GluN2B tyrosine phosphorylation was upregulated in SOD1G93A. Tyrosine phosphorylation of GluN2B subunits regulates the NMDAR function and the recruitment of downstream signaling molecules. Indeed, we found that proline-rich tyrosine kinase 2 (Pyk2) and ERK1/2 kinase are upregulated in SOD1G93A mice. These results point out an involvement of tyrosine kinases and phosphatases in the pathogenesis of ALS.

scholarly journals A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling

2016 ◽  
Vol 91 (2) ◽  
Author(s):  
Deborah Denis ◽  
Cecile Rouleau ◽  
Brian S. Schaffhausen
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
T Antigen ◽  
Pi3 Kinase ◽  
Binding Motif ◽  
Wild Type ◽  
Pi3k Activity ◽  
Murine Polyomavirus ◽  
Middle T Antigen

ABSTRACT Middle T antigen (MT), the principal oncoprotein of murine polyomavirus, transforms by association with cellular proteins. Protein phosphatase 2A (PP2A), YAP, Src family tyrosine kinases, Shc, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLCγ1) have all been implicated in MT transformation. Mutant dl1015, with deletion of residues 338 to 347 in the C-terminal region, has been an enigma, because the basis for its transformation defect has not been apparent. This work probes the dl1015 region of MT. Because the region is proline rich, the hypothesis that it targets Src homology domain 3 (SH3) domains was tested, but mutation of the putative SH3 binding motif did not affect transformation. During this work, two point mutants, W348R and E349K, were identified as transformation defective. Extensive analysis of the E349K mutant is described here. Similar to wild-type MT, the E349K mutant associates with PP2A, YAP, tyrosine kinases, Shc, PI3 kinase, and PLCγ1. The E349K mutant was examined to determine the mechanism for its transformation defect. Assays of cell localization and membrane targeting showed no obvious difference in localization. Src association was normal as assayed by in vitro kinase and MT phosphopeptide mapping. Shc activation was confirmed by its tyrosine phosphorylation. Association of type 1 PI3K with MT was demonstrated by coimmunoprecipitation, showing both PI3K subunits and in vitro activity. Nonetheless, expression of the mutants failed to lead to the activation of two known downstream targets of PI3K, Akt and Rac-1. Strikingly, despite normal association of the E349K mutant with PI3K, cells expressing the mutant failed to elevate phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in mutant-expressing cells. These results indicate a novel unsuspected aspect to PI3K control. IMPORTANCE The gene coding for middle T antigen (MT) is the murine polyomavirus oncogene most responsible for tumor formation. Its study has a history of uncovering novel aspects of mammalian cell regulation. The importance of PI3K activity and tyrosine phosphorylation are two examples of insights coming from MT. This study describes new mutants unable to transform like the wild type that point to novel regulation of PI3K signaling. Previous mutants were defective in PI3K because they failed to bind the enzyme and bring the activity to the membrane. These mutants recruit PI3K activity like the wild type, but fail to elevate the cellular level of PIP3, the product used to signal downstream of PI3K. As a result, they fail to activate either Akt or Rac1, explaining the transformation defect.

Optimisation of EGFR TKI efficiency in the therapeutic scheme of EGFR wild-type lung cancer.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e18532-e18532
Author(s):  
Mathilde Cabart ◽  
Judith Raimbourg ◽  
Lisenn Lalier ◽  
Jaafar Bennouna ◽  
Francois Vallette
Keyword(s):  
Lung Cancer ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Wild Type ◽  
Short Term ◽  
Egfr Ligands ◽  
Egfr Mutated ◽  
Egfr Tki

e18532 Background: EGFR tyrosine kinase inhibitors (EGFR TKI) have improved the therapeutic care of lung cancer patients but only a small sub-population of patients, namely those harboring EGFR-mutated tumors, benefit from this therapy. The observation that EGFR TKI enhance prognosis and quality of life in all patients when used as second line or maintenance treatment impelled us into the search of potential markers of the optimal introduction kinetics of EGFR TKI in the therapeutic scheme. Methods: We used lung cancer cell lines harboring either wild-type or mutated EGFR (NCI-H1650, NCI-H1975) to study the consequences of cisplatin treatment in vitro on the consecutive sensitivity to erlotinib. Results: Sub-lethal cisplatin pretreatment (3µM) enhances erlotinib toxicity in EGFR wild-type, but not EGFR mutated cells (A549 IC50 drops from 28 to 15µM for short-term or 12µM for long-term exposure). This correlates with EGFR activation following short-term or prolonged cisplatin treatment through the secretion of EGFR ligands. This activation of EGFR is concomitant to the decrease in other receptor tyrosine kinases phosphorylation including Met. Conclusions: The sensitivity of EGFR wild-type lung cancer cells to erlotinib in vitro is enhanced by cisplatin pretreatment. We identified potential markers of this sensitization, namely EGFR ligands, which serum level might be predicitive of the optimal efficiency of EGFR TKI. In vivo validation of these markers is under investigation. The concomitant decrease in other receptor tyrosine kinases phosphorylation suggests that the targeting of other receptor tyrosine kinases might potentiate EGFR TKI efficiency.

Role of Fc receptor γ-chain in platelet glycoprotein Ib–mediated signaling

Blood ◽  
2001 ◽  
Vol 97 (12) ◽  
pp. 3836-3845 ◽  
Author(s):  
Yi Wu ◽  
Katsue Suzuki-Inoue ◽  
Kaneo Satoh ◽  
Naoki Asazuma ◽  
Yutaka Yatomi ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Calcium Release ◽  
Human Platelets ◽  
Fc Receptor ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Glycoprotein Ib ◽  
Von Willebrand ◽  
Willebrand Factor

Interaction between von Willebrand factor (vWF) and glycoprotein Ib (GPIb) stimulates tyrosine kinases and subsequent tyrosine phosphorylation events in human platelets. This study found that the combination of vWF and botrocetin, by interacting with GPIb, induced tyrosine phosphorylation of Fc receptor γ-chain (FcR γ-chain), Syk, linker for activation of T cells (LAT), and phospholipase C γ2 (PLCγ2). Pretreatment of platelets with 10 μM PP1 completely inhibited these tyrosine phosphorylation events. On GPIb stimulation, Src and Lyn formed a complex with FcR γ-chain and Syk, suggesting that Src and Lyn are involved in FcR γ-chain tyrosine phosphorylation and downstream signals. In spite of the PLCγ2 tyrosine phosphorylation, however, there was no intracellular calcium release and inositol 1,4,5-trisphosphate production. In Brij 35 lysates, FcR γ-chain was found to constitutively associate with GPIb. The number of GPIb expressed on FcR γ-chain–deficient platelets was comparable to that of the wild-type, as assessed by flow cytometry. However, tyrosine phosphorylation of Syk, LAT, and PLCγ2 in response to vWF plus botrocetin was significantly suppressed, suggesting that FcR γ-chain mediates activation signals related to GPIb. Compared with the aggregation response of wild-type platelets, that of FcR γ-chain–deficient platelets in response to vWF plus botrocetin was impaired, implying that FcR γ-chain is required for the full activation of platelets mediated by GPIb.

scholarly journals Constitutive secretion of serum albumin requires reversible protein tyrosine phosphorylation events in trans-Golgi

2005 ◽  
Vol 289 (3) ◽  
pp. C748-C756 ◽  
Author(s):  
Rachel J. Webb ◽  
Jacob D. Judah ◽  
Lee-Chiang Lo ◽  
Geraint M. H. Thomas
Keyword(s):  
Serum Albumin ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Protein Tyrosine Phosphorylation ◽  
Albumin Secretion ◽  
Protein Tyrosine ◽  
Constitutive Secretion

Serum albumin secretion from rat hepatocytes proceeds via the constitutive pathway. Although much is known about the role of protein tyrosine phosphorylation in regulated secretion, nothing is known about its function in the constitutive process. Here we show that albumin secretion is inhibited by the tyrosine kinase inhibitor genistein but relatively insensitive to subtype-selective inhibitors or treatments. Secretion is also blocked in a physiologically identical manner by the tyrosine phosphatase inhibitors pervanadate and bisperoxo(1,10-phenanthroline)-oxovanadate. Inhibition of either the kinase(s) or phosphatase(s) leads to the accumulation of albumin between the trans-Golgi and the plasma membrane, whereas the immediate precursor proalbumin builds up in a proximal compartment. The trans-Golgi marker TGN38 is rapidly dispersed under conditions that inhibit tyrosine phosphatase action, whereas the distribution of the cis-Golgi marker GM130 is insensitive to genistein or pervanadate. By using a specifically reactive biotinylation probe, we detected protein tyrosine phosphatases in highly purified rat liver Golgi membranes. These membranes also contain both endogenous tyrosine kinases and their substrates, indicating that enzymes and substrates for reversible tyrosine phosphorylation are normal membrane-resident components of this trafficking compartment. In the absence of perturbation of actin filaments and microtubules, we conclude that reversible protein tyrosine phosphorylation in the trans-Golgi network is essential for albumin secretion and propose that the constitutive secretion of albumin is in fact a regulated process.

scholarly journals Activation of PTHrP-cAMP-CREB1 signaling following p53 loss is essential for osteosarcoma initiation and maintenance

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Mannu K Walia ◽  
Patricia MW Ho ◽  
Scott Taylor ◽  
Alvin JM Ng ◽  
Ankita Gupte ◽  
...  
Keyword(s):  
Human Cancer ◽  
Point Mutations ◽  
Therapeutic Targets ◽  
Complete Loss ◽  
Osteoblastic Cells ◽  
Normal Cells ◽  
P53 Pathway ◽  
Common Cancer ◽  
Frequent Event ◽  
Camp Levels

Mutations in the P53 pathway are a hallmark of human cancer. The identification of pathways upon which p53-deficient cells depend could reveal therapeutic targets that may spare normal cells with intact p53. In contrast to P53 point mutations in other cancer, complete loss of P53 is a frequent event in osteosarcoma (OS), the most common cancer of bone. The consequences of p53 loss for osteoblastic cells and OS development are poorly understood. Here we use murine OS models to demonstrate that elevated Pthlh (Pthrp), cAMP levels and signalling via CREB1 are characteristic of both p53-deficient osteoblasts and OS. Normal osteoblasts survive depletion of both PTHrP and CREB1. In contrast, p53-deficient osteoblasts and OS depend upon continuous activation of this pathway and undergo proliferation arrest and apoptosis in the absence of PTHrP or CREB1. Our results identify the PTHrP-cAMP-CREB1 axis as an attractive pathway for therapeutic inhibition in OS.

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