scholarly journals Protein tyrosine phosphorylation in pancreatic acini: differential effects of VIP and CCK

1997 ◽  
Vol 273 (6) ◽  
pp. G1226-G1232
Author(s):  
Manfred P. Lutz ◽  
Albrecht Piiper ◽  
Herbert Y. Gaisano ◽  
Danuta Stryjek-Kaminska ◽  
Stefan Zeuzem ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Adenylyl Cyclase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Electrophoretic Separation ◽  
Direct Stimulation ◽  
Pancreatic Acini ◽  
Dependent Protein ◽  
Tyrphostin 23

Cholecystokinin (CCK) and vasoactive intestinal peptide (VIP) stimulate enzyme secretion from pancreatic acini by binding to heptahelical receptors without intrinsic tyrosine kinase activity. Signal transduction by the CCK receptor involves activation of phospholipase C by Gq proteins and activation of tyrosine kinases, whereas occupation of VIP receptors stimulates adenylyl cyclase through binding to Gs proteins. Here, we use electrophoretic separation of cellular proteins and antiphosphotyrosine immunoblotting to demonstrate a VIP-stimulated rapid and dose-dependent increase in tyrosine phosphorylation of proteins migrating at 130, 115, and 93 kDa in freshly isolated rat pancreatic acini. Phosphorylation of these proteins was increased after direct stimulation of adenylyl cyclase or the adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase with forskolin or dibutyryl cAMP and was inhibited by the tyrosine kinase inhibitors genistein or tyrphostin 23. Compared with VIP, CCK stimulated tyrosine phosphorylation of additional proteins migrating at 60, 66, and 72/78 kDa. Using two-dimensional electrophoretic separation or immunoprecipitation, the 72/78-kDa phosphoprotein was identified as paxillin. We propose that paxillin might be involved in CCK- but not in VIP-induced exocytosis.

[3H]taurine andd-[3H]aspartate release from astrocyte cultures are differently regulated by tyrosine kinases

1999 ◽  
Vol 276 (5) ◽  
pp. C1226-C1230 ◽  
Author(s):  
Alexander A. Mongin ◽  
Jyoti M. Reddi ◽  
Carol Charniga ◽  
Harold K. Kimelberg
Keyword(s):  
Amino Acids ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Mammalian Brain ◽  
Dependent Manner ◽  
Anion Channels ◽  
Primary Astrocyte ◽  
Tyrphostin 23

Volume-dependent anion channels permeable for Cl− and amino acids are thought to play an important role in the homeostasis of cell volume. Astrocytes are the main cell type in the mammalian brain showing volume perturbations under physiological and pathophysiological conditions. We investigated the involvement of tyrosine phosphorylation in hyposmotic medium-induced [3H]taurine andd-[3H]aspartate release from primary astrocyte cultures. The tyrosine kinase inhibitors tyrphostin 23 and tyrphostin A51 partially suppressed the volume-dependent release of [3H]taurine in a dose-dependent manner with half-maximal effects at ∼40 and 1 μM, respectively. In contrast, the release ofd-[3H]aspartate was not significantly affected by these agents in the same concentration range. The inactive analog tyrphostin 1 had no significant effect on the release of both amino acids. The data obtained suggest the existence of at least two volume-dependent anion channels permeable to amino acids in astrocyte cultures. One of these channels is permeable to taurine and is under the control of tyrosine kinase(s). The other is permeable to both taurine and aspartate, but its volume-dependent regulation does not require tyrosine phosphorylation.

Role for tyrosine kinases in carbachol-regulated Ca entry into colonic epithelial cells

1995 ◽  
Vol 268 (1) ◽  
pp. C154-C161 ◽  
Author(s):  
G. Bischof ◽  
B. Illek ◽  
W. W. Reenstra ◽  
T. E. Machen
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Divalent Cations ◽  
Tyrosine Phosphatase ◽  
Inhibitory Effect ◽  
Colonic Epithelial Cells

We studied a possible role of tyrosine kinases in the regulation of Ca entry into colonic epithelial cells HT-29/B6 using digital image processing of fura 2 fluorescence. Both carbachol and thapsigargin increased Ca entry to a similar extent and Ca influx was reduced by the tyrosine kinase inhibitor genistein (50 microM). Further experiments were performed in solutions containing 95 mM K to depolarize the membrane potential, and the effects of different inhibitors on influx of Ca, Mn, and Ba were compared. Genistein, but not the inactive analogue daidzein nor the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine, decreased entry of all three divalent cations by 47-59%. In high-K solutions, carbachol or thapsigargin both caused intracellular Ca to increase to a plateau of 223 +/- 19 nM. This plateau was reduced by the tyrosine kinase inhibitors genistein (to 95 +/- 8 nM), lavendustin A (to 155 +/- 17 nM), and methyl-2,5-dihydroxycinnamate (to 39 +/- 3 nM). Orthovanadate, a protein tyrosine phosphatase inhibitor, prevented the inhibitory effect of genistein. Ca pumping was unaffected by genistein. Carbachol increased tyrosine phosphorylation (immunoblots with anti-phosphotyrosine antibodies) of 110-, 75-, and 70-kDa proteins, and this phosphorylation was inhibited by genistein. We conclude that carbachol and thapsigargin increase Ca entry, and tyrosine phosphorylation of some key proteins may be important for regulating this pathway.

Thrombin Mediated Tyrosine Phosphorylation of PKCδ in Human Platelets Occurs in a Calcium- and Src Family Tyrosine Kinase- Dependent Manner.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3541-3541
Author(s):  
Swaminathan Murugappan ◽  
Haripriya Shankar ◽  
Satya Kunapuli
Keyword(s):  
Tyrosine Kinase ◽  
Intracellular Calcium ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Human Platelets ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Glycoprotein Vi ◽  
Pkc Isoforms

Abstract Protein kinase C (PKC)-δ is a novel PKC that has been shown to be tyrosine phosphorylated upon stimulation with agonists in platelets. Tyrosine phosphorylation of PKCδ has been shown to occur in a Fyn-dependent manner downstream of glycoprotein VI (GPVI) signaling in platelets. Although thrombin causes tyrosine phosphorylation of PKCδ in platelets, the mechanism of this event is not elucidated. In this study, we investigated whether G-protein signaling pathways utilize similar pathways as GPVI in tyrosine phosphorylation of PKCδ. Protease activated receptor (PAR) -1 selective peptide, SFLLRN and PAR - 4 selective peptide, AYPGKF caused a time- and concentration-dependent increase in tyrosine phosphorylation of PKCδ in human platelets. However, AYPGKF failed to cause tyrosine phosphorylation of PKCδ in Gq-deficient mouse platelets. Both U73122, a phospholipase C (PLC) inhibitor, and dimethyl-BAPTA, an intracellular calcium chelator, inhibited the tyrosine phosphorylation of PKCδ downstream of the PAR activation suggesting a role for Gq/PLC pathways and intracellular calcium in mediating this event. Inhibition of PKC isoforms using GF109203X potentiated the tyrosine phosphorylation of PKCδ. The Src family tyrosine kinase inhibitors, PP1 and PP2 inhibited the tyrosine phosphorylation of PKCδ suggesting a role for Src family tyrosine kinase members in this event. We also found that both Lyn and Src are physically associated with PKCδ in a constitutive manner in platelets. Finally we found that there was a time-dependent activation of Src following activation of platelets with thrombin. Thus, the precomplexed Src and Lyn tyrosine kinases get activated following PAR stimulation resulting in the tyrosine phosphorylation of PKCδ. All these data indicate that tyrosine phosphorylation of PKCδ downstream of thrombin occurs in a calcium- and Src-family kinase dependent manner in human platelets.

Are tyrosine kinases involved in mediating contraction-stimulated muscle glucose transport?

2006 ◽  
Vol 290 (1) ◽  
pp. E123-E128 ◽  
Author(s):  
David C. Wright ◽  
Paige C. Geiger ◽  
Dong-Ho Han ◽  
John O. Holloszy
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Tyrosine Kinase ◽  
Glucose Transport ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Muscle Contractions ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Muscle contractions and insulin stimulate glucose transport into muscle by separate pathways. The contraction-mediated increase in glucose transport is mediated by two mechanisms, one involves the activation of 5′-AMP-activated protein kinase (AMPK) and the other involves the activation of calcium/calmodulin-dependent protein kinase II (CAMKII). The steps leading from the activation of AMPK and CAMKII to the translocation of GLUT4 to the cell surface have not been identified. Studies with the use of the tyrosine kinase inhibitor genistein suggest that one or more tyrosine kinases could be involved in contraction-stimulated glucose transport. The purpose of the present study was to determine the involvement of tyrosine kinases in contraction-stimulated glucose transport in rat soleus and epitrochlearis muscles. Contraction-stimulated glucose transport was completely prevented by pretreatment with genistein (100 μM) and the related compound butein (100 μM). However, the structurally distinct tyrosine kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4-d]pyridine and herbimycin did not reduce contraction-stimulated glucose transport. Furthermore, genistein and butein inhibited glucose transport even when muscles were exposed to these compounds after being stimulated to contract. Muscle contractions did not result in increases in tyrosine phosphorylation of proteins such as proline-rich tyrosine kinase and SRC. These results provide evidence that tyrosine kinases do not mediate contraction-stimulated glucose transport and that the inhibitory effects of genistein on glucose transport result from direct inhibition of the glucose transporters at the cell surface.

scholarly journals Tyrosine phosphorylation is involved in reorganization of the actin cytoskeleton in response to serum or LPA stimulation

1994 ◽  
Vol 107 (12) ◽  
pp. 3643-3654 ◽  
Author(s):  
M. Chrzanowska-Wodnicka ◽  
K. Burridge
Keyword(s):  
Tyrosine Kinase ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinase Inhibitors ◽  
Lysophosphatidic Acid ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Focal Adhesions ◽  
Protein Tyrosine Phosphatases ◽  
Stress Fibers

Tyrosine phosphorylation is known to regulate the formation of focal adhesions in cells adhering to extracellular matrix (ECM). We have investigated the possible involvement of tyrosine phosphorylation and the focal adhesion kinase (FAK) in the cytoskeletal changes induced by serum or lysophosphatidic acid (LPA) in quiescent Swiss 3T3 fibroblasts. As shown previously by others, quiescent cells stimulated with serum or LPA reveal a rapid reappearance of focal adhesions and stress fibers. Here we show that this is accompanied by an increase in phosphotyrosine in focal adhesions and specifically an increase in the tyrosine phosphorylation of FAK. The LPA-stimulated reappearance of focal adhesions and stress fibers is blocked by inhibitors of phospholipase C but not by pertussis toxin (PTX), indicating that this LPA signaling pathway is mediated by phospholipase C activation and does not involve PTX-sensitive G proteins. In the absence of serum or LPA, these cytoskeletal effects and the tyrosine phosphorylation of FAK can be mimicked by sodium orthovanadate in conjunction with hydrogen peroxide, agents that inhibit protein tyrosine phosphatases and thereby elevate levels of phosphotyrosine. Two tyrosine kinase inhibitors, erbstatin and genistein block both the serum-induced tyrosine phosphorylation of FAK and the assembly of focal adhesions and stress fibers. Two other tyrosine kinase inhibitors, tyrphostins 47 and 25, previously shown to inhibit FAK, failed to prevent FAK phosphorylation or the reassembly of focal adhesions and stress fibers in response to serum. However, these inhibitors did prevent FAK phosphorylation and cytoskeletal assembly in response to lysophosphatidic acid (LPA), one component of serum previously shown to stimulate assembly of focal adhesions and stress fibers. Our findings suggest that the response to serum is complex and that although FAK phosphorylation is important, other tyrosine kinases may also be involved.

scholarly journals Identification of tyrosine kinase inhibitors that halt Plasmodium falciparum parasitemia

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242372
Author(s):  
Kristina Kesely ◽  
Panae Noomuna ◽  
Michal Vieth ◽  
Philip Hipskind ◽  
Kasturi Haldar ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinase Inhibitors ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Antimalarial Activity ◽  
Band 3 ◽  
Parasite Egress

Although current malaria therapies inhibit pathways encoded in the parasite’s genome, we have looked for anti-malaria drugs that can target an erythrocyte component because development of drug resistance might be suppressed if the parasite cannot mutate the drug’s target. In search for such erythrocyte targets, we noted that human erythrocytes express tyrosine kinases, whereas the Plasmodium falciparum genome encodes no obvious tyrosine kinases. We therefore screened a library of tyrosine kinase inhibitors from Eli Lilly and Co. in a search for inhibitors with possible antimalarial activity. We report that although most tyrosine kinase inhibitors exerted no effect on parasite survival, a subset of tyrosine kinase inhibitors displayed potent anti-malarial activity. Moreover, all inhibitors found to block tyrosine phosphorylation of band 3 specifically suppressed P. falciparum survival at the parasite egress stage of its intra-erythrocyte life cycle. Conversely, tyrosine kinase inhibitors that failed to block band 3 tyrosine phosphorylation but still terminated the parasitemia were observed to halt parasite proliferation at other stages of the parasite’s life cycle. Taken together these results suggest that certain erythrocyte tyrosine kinases may be important to P. falciparum maturation and that inhibitors that block these kinases may contribute to novel therapies for P. falciparum malaria.

scholarly journals Tyrosine phosphorylation and association of Syk with FcγRII in monocytic THP-1 cells

1995 ◽  
Vol 305 (2) ◽  
pp. 669-674 ◽  
Author(s):  
S Ghazizadeh ◽  
J B Bolen ◽  
H B Fleit
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Immune Recognition ◽  
High Affinity ◽  
Gamma Receptor ◽  
High Affinity Ige Receptor

Although the cytoplasmic portion of the low-affinity receptor for immunoglobulin G, Fc gamma RII, does not contain a kinase domain, rapid tyrosine phosphorylation of intracellular substrates occurs in response to aggregation of the receptor. The use of specific tyrosine kinase inhibitors has suggested that these phosphorylations are required for subsequent cellular responses. We previously demonstrated the coprecipitation of a tyrosine kinase activity with Fc gamma RII, suggesting that non-receptor tyrosine kinases might associate with the cytoplasmic domain of Fc gamma RII. Anti-receptor immune complex kinase assays revealed the coprecipitation of several phosphoproteins, most notably p56/53lyn, an Src-family protein tyrosine kinase (PTK), and a 72 kDa phosphoprotein. Here we identify the 72 kDa Fc gamma RII-associated protein as p72syk (Syk), a member of a newly described family of non-receptor PTKs. A rapid and transient tyrosine phosphorylation of Syk was observed following Fc gamma RII activation. Syk was also tyrosyl-phosphorylated following aggregation of the high-affinity Fc gamma receptor, Fc gamma RI. The Fc gamma RI activation did not result in association of Syk with Fc gamma RII, implying that distinct pools of Syk are activated upon aggregation of each receptor in a localized manner. These results demonstrate a physical association between Syk and Fc gamma RII and suggest that the molecules involved in Fc gamma RII signalling are very similar to the ones utilized by multichain immune recognition receptors such as the B-cell antigen receptor and the high-affinity IgE receptor.

scholarly journals Novel Receptor Tyrosine Kinase Pathway Inhibitors for Targeted Radionuclide Therapy of Glioblastoma

2021 ◽  
Vol 14 (7) ◽  
pp. 626
Author(s):  
Julie Bolcaen ◽  
Shankari Nair ◽  
Cathryn H. S. Driver ◽  
Tebatso M. G. Boshomane ◽  
Thomas Ebenhan ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Radionuclide Therapy ◽  
Kinase Inhibitors ◽  
Nuclear Imaging ◽  
Therapy Resistance ◽  
Targeted Radionuclide Therapy ◽  
Therapy Strategy ◽  
Dismal Prognosis

Glioblastoma (GB) remains the most fatal brain tumor characterized by a high infiltration rate and treatment resistance. Overexpression and/or mutation of receptor tyrosine kinases is common in GB, which subsequently leads to the activation of many downstream pathways that have a critical impact on tumor progression and therapy resistance. Therefore, receptor tyrosine kinase inhibitors (RTKIs) have been investigated to improve the dismal prognosis of GB in an effort to evolve into a personalized targeted therapy strategy with a better treatment outcome. Numerous RTKIs have been approved in the clinic and several radiopharmaceuticals are part of (pre)clinical trials as a non-invasive method to identify patients who could benefit from RTKI. The latter opens up the scope for theranostic applications. In this review, the present status of RTKIs for the treatment, nuclear imaging and targeted radionuclide therapy of GB is presented. The focus will be on seven tyrosine kinase receptors, based on their central role in GB: EGFR, VEGFR, MET, PDGFR, FGFR, Eph receptor and IGF1R. Finally, by way of analyzing structural and physiological characteristics of the TKIs with promising clinical trial results, four small molecule RTKIs were selected based on their potential to become new therapeutic GB radiopharmaceuticals.

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 Na+/H+ exchange activity during phagocytosis in human neutrophils: role of Fcgamma receptors and tyrosine kinases.

1996 ◽  
Vol 132 (6) ◽  
pp. 1037-1052 ◽  
Author(s):  
T Fukushima ◽  
T K Waddell ◽  
S Grinstein ◽  
G G Goss ◽  
J Orlowski ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Plasma Membranes ◽  
Kinase Inhibitors ◽  
Human Neutrophils ◽  
Cross Linking ◽  
Fcgamma Receptors ◽  
Beta2 Integrins ◽  
Cytosolic Acidification

In neutrophils, binding and phagocytosis facilitate subsequent intracellular killing of microorganisms. Activity of Na+/H+ exchangers (NHEs) participates in these events, especially in regulation of intracellular pH (pHi) by compensating for the H+ load generated by the respiratory burst. Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutrophils. The purpose of this study was to identify which NHE(s) are expressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis. Exposure of cells to the phagocytic stimulus opsonized zymosan (OpZ) induced a transient cytosolic acidification followed by a prolonged alkalinization. The latter was inhibited in Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H+ exchange. Reverse transcriptase PCR and cDNA sequencing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed. Immunoblotting of purified plasma membranes with isoform-specific antibodies confirmed the presence of NHE-1 protein in neutrophils. Since phagocytosis involves Fcgamma (FcgammaR) and complement receptors such as CR3 (a beta2 integrin) which are linked to pathways involving alterations in intracellular [Ca2+]i and tyrosine phosphorylation, we studied these pathways in relation to activation of NHE-1. Cross-linking of surface bound antibodies (mAb) directed against FcgammaRs (FcgammaRII > FcgammaRIII) but not beta2 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-beta2 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE-1 activation involved cooperation between integrins and FcgammaRs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcgammaR cross-linking suggesting that tyrosine phosphorylation was involved in NHE-I activation. An increase in [Ca2+]i was not required for NHE-1 activation because neither removal of extracellular Ca2+ nor buffering of changes in [Ca2+]i inhibited alkalinization after OpZ or Fc-gammaR cross-linking. In summary, Fc-gammaRs and beta2 integrins cooperate in activation of NHE-1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation.

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