Remodeling of cytoskeleton and triads following activation of v-Src tyrosine kinase in quail myotubes

1996 ◽  
Vol 109 (6) ◽  
pp. 1335-1346
Author(s):  
L. Castellani ◽  
M. Reedy ◽  
J.A. Airey ◽  
R. Gallo ◽  
M.T. Ciotti ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Tyrosine Kinase ◽  
Intermediate Filaments ◽  
Temperature Sensitive ◽  
Src Tyrosine Kinase ◽  
Kinase Activation ◽  
Maturation In Vitro ◽  
Immunofluorescence Labeling ◽  
Triad Junctions

To study the cellular signals underlying the regulatory mechanisms involved in maintenance of sarcomeric integrity, we have used quail skeletal muscle cells that reach a high degree of structural maturation in vitro, and also express a temperature-sensitive mutant of the v-Src tyrosine kinase that allows the control of differentiation in a reversible manner. By immunofluorescence and electron microscopy we show that v-Src activity in myotubes leads to an extensive cellular remodeling which affects components of the sarcomeres, the cytoskeleton network and the triad junctions. We have previously shown that activation of v-Src causes a selective dismantling of the I-Z-I segments coupled to the formation of aggregates of sarcomeric actin, alpha-actinin and vinculin, called actin bodies. We now show that intermediate filaments do not participate in the formation of actin bodies, while talin, a component of costameres, does. The I-Z-I segments are completely dismantled within 24 hours of v-Src activity, but the A-bands persist for a longer time, implying distinct pathways for the turnover of sarcomeric subdomains. Immunofluorescence labeling of markers of the triad junctions demonstrates that the localization of the alpha 1 subunit of the dihydropyridine receptor is disrupted earlier than that of the ryanodine receptor after tyrosine kinase activation. Furthermore, the location of junctional sarcoplasmic reticulum and transverse tubule membranes is maintained in myotubes in which the I-Z-I have been removed and the regular disposition of the intermediate filaments is disrupted, supporting a role for sarcoplasmic reticulum in the proper positioning of triad junctions. Altogether these results point to a tyrosine kinase signaling cascade as a mechanism for selectively destabilizing sarcomere subdomains and their tethering to the cytoskeleton and the sarcolemma.

scholarly journals Dihydropyridine receptor alpha subunits in normal and dysgenic muscle in vitro: expression of alpha 1 is required for proper targeting and distribution of alpha 2.

1991 ◽  
Vol 115 (5) ◽  
pp. 1345-1356 ◽  
Author(s):  
B E Flucher ◽  
J L Phillips ◽  
J A Powell
Keyword(s):  
Muscle Cells ◽  
Distribution Patterns ◽  
Receptor Alpha ◽  
Alpha Subunits ◽  
Nuclear Domain ◽  
T Tubules ◽  
Immunofluorescence Labeling ◽  
T System ◽  
Triad Junctions

We have studied the subcellular distribution of the alpha 1 and alpha 2 subunits of the skeletal muscle dihydropyridine (DHP) receptor with immunofluorescence labeling of normal and dysgenic (mdg) muscle in culture. In normal myotubes both alpha subunits were localized in clusters associated with the T-tubule membranes of longitudinally as well as transversely oriented T-tubules. The DHP receptor-rich domains may represent the sites where triad junctions with the sarcoplasmic reticulum are being formed. In cultures from dysgenic muscle the alpha 1 subunit was undetectable and the distribution patterns of the alpha 2 subunit were abnormal. The alpha subunit did not form clusters nor was it discretely localized in the T-tubule system. Instead, alpha 2 was found diffusely distributed in parts of the T-system, in structures in the perinuclear region and in the plasma membrane. These results suggest that an interaction between the two alpha subunits is required for the normal distribution of the alpha 2 subunit in the T-tubule membranes. Spontaneous fusion of normal non-muscle cells with dysgenic myotubes resulted in a regional expression of the alpha 1 polypeptide near the foreign nuclei, thus defining the nuclear domain of a T-tubule membrane protein in multi-nucleated muscle cells. Furthermore, the normal intracellular distribution of the alpha 2 polypeptide was restored in domains containing a foreign "rescue" nucleus; this supports the idea that direct interactions between the DHP receptor alpha 1 and alpha 2 subunits are involved in the organization of the junctional T-tubule membranes.

scholarly journals Oligomerization of the ABL tyrosine kinase by the Ets protein TEL in human leukemia.

1996 ◽  
Vol 16 (8) ◽  
pp. 4107-4116 ◽  
Author(s):  
T R Golub ◽  
A Goga ◽  
G F Barker ◽  
D E Afar ◽  
J McLaughlin ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Bone Marrow Cells ◽  
Chromosomal Translocation ◽  
Human Leukemia ◽  
Kinase Activation ◽  
Murine Bone Marrow ◽  
Helix Loop Helix ◽  
Ets Proteins ◽  
Abl Tyrosine Kinase

TEL is a member of the Ets family of transcription factors which are frequently rearranged in human leukemia. The mechanism of TEL-mediated transformation, however, is unknown. We report the cloning and characterization of a chromosomal translocation associated with acute myeloid leukemia which fuses TEL to the ABL tyrosine kinase. The TEL-ABL fusion confers growth factor-independent growth to the marine hematopoietic cell line Ba/F3 and transforms Rat-1 fibroblasts and primary murine bone marrow cells. TEL-ABL is constitutively tyrosine phosphorylated and localizes to the cytoskeleton. A TEL-ABL mutant containing an ABL kinase-inactivating mutation is not constitutively phosphorylated and is nontransforming but retains cytoskeletal localization. However, constitutive phosphorylation, cytoskeletal localization, and transformation are all dependent upon a highly conserved region of TEL termed the helix-loop-helix (HLH) domain. TEL-ABL formed HLH-dependent homo-oligomers in vitro, a process critical for tyrosine kinase activation. These experiments suggest that oligomerization of TEL-ABL mediated by the TEL HLH domain is required for tyrosine kinase activation, cytoskeletal localization, and transformation. These data also suggest that oligomerization of Ets proteins through the highly conserved HLH domain may represent a previously unrecognized phenomenon.

scholarly journals Mammary tumors expressing the neu proto-oncogene possess elevated c-Src tyrosine kinase activity.

1994 ◽  
Vol 14 (1) ◽  
pp. 735-743 ◽  
Author(s):  
S K Muthuswamy ◽  
P M Siegel ◽  
D L Dankort ◽  
M A Webster ◽  
W J Muller
Keyword(s):  
Signal Transduction ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Specific Activity ◽  
Fusion Gene ◽  
Mammary Tumorigenesis ◽  
Mammary Tumors ◽  
Tyrosine Kinase Activity ◽  
Src Tyrosine Kinase

Amplification and overexpression of the neu (c-erbB2) proto-oncogene has been implicated in the pathogenesis of 20 to 30% of human breast cancers. Although the activation of Neu receptor tyrosine kinase appears to be a pivotal step during mammary tumorigenesis, the mechanism by which Neu signals cell proliferation is unclear. Molecules bearing a domain shared by the c-Src proto-oncogene (Src homology 2) are thought to be involved in signal transduction from activated receptor tyrosine kinases such as Neu. To test whether c-Src was implicated in Neu-mediated signal transduction, we measured the activity of the c-Src tyrosine kinase in tissue extracts from either mammary tumors or adjacent mammary epithelium derived from transgenic mice expressing a mouse mammary tumor virus promoter/enhancer/unactivated neu fusion gene. The Neu-induced mammary tumors possessed six- to eightfold-higher c-Src kinase activity than the adjacent epithelium. The increase in c-Src tyrosine kinase activity was not due to an increase in the levels of c-Src but rather was a result of the elevation of its specific activity. Moreover, activation of c-Src was correlated with its ability to complex tyrosine-phosphorylated Neu both in vitro and in vivo. Together, these observations suggest that activation of the c-Src tyrosine kinase during mammary tumorigenesis may occur through a direct interaction with activated Neu.

scholarly journals Tyrosine phosphorylation of mammalian RNA polymerase II carboxyl-terminal domain

1993 ◽  
Vol 90 (23) ◽  
pp. 11167-11171 ◽  
Author(s):  
R Baskaran ◽  
M E Dahmus ◽  
J Y Wang
Keyword(s):  
Tyrosine Kinase ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Tandem Repeats ◽  
Consensus Sequence ◽  
Src Tyrosine Kinase ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Phosphorylation of the CTD occurs during formation of the initiation complex and is correlated with the transition from complex assembly to elongation. Previously, serine and threonine residues within the CTD have been shown to be modified by the addition of phosphate and by the addition of O-linked GlcNAc. Our results establish that the CTD is also modified in vivo by phosphorylation on tyrosine. Furthermore, a nuclear tyrosine kinase encoded by the c-abl protooncogene phosphorylates the CTD to a high stoichiometry in vitro. Under conditions of maximum phosphorylation, approximately 30 mol of phosphate are incorporated per mol of CTD. The observation that the CTD is not phosphorylated by c-Src tyrosine kinase under identical conditions indicates that the CTD is not a substrate of all tyrosine kinases. Phosphorylation of tyrosine residues within the CTD may modulate the interaction of RNA polymerase II with the preinitiation complex and, hence, may be important in regulating gene expression.

Prolactin induced tyrosine phosphorylation of p59fyn may mediate phosphatidylinositol 3-kinase activation in Nb2 cells

1997 ◽  
Vol 19 (3) ◽  
pp. 347-350 ◽  
Author(s):  
KA al-Sakkaf ◽  
PR Dobson ◽  
BL Brown
Keyword(s):  
Tyrosine Phosphorylation ◽  
Pi3 Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Src Tyrosine Kinase ◽  
Kinase Activation ◽  
Nb2 Cells ◽  
Co Precipitation ◽  
Dose Dependent ◽  
Phosphatidylinositol 3

Our previous studies indicated that PI3-kinase is involved in prolactin (PRL) signalling. We have now examined the involvement of the src tyrosine kinase, fyn, in PRL-induced the activation of PI3-kinase in the rat lymphoma cell line, Nb2. Cells were stimulated with increasing doses of PRL, lysed and immunoprecipitated with anti-fyn specific antibody. Then PI3-kinase activity was measured as the increase in the phosphorylation of phosphatidylinositol to phosphatidylinositol 3-phosphate separated by TLC. Our data indicated that, in PRL treated cells, co-precipitation of PI3-kinase with anti-fyn antiserum led to time and dose-dependent activation of PI3-kinase in vitro and that this activation was blocked by the addition of LY294002. However, LY294002 appeared to have no effect on fyn autophosphorylation. Furthermore, the physical association of PI3-kinase with fyn was confirmed by Western blot analysis employing the same specific antisera. These data provide evidence that PRL-induced activation of PI3-kinase may be mediated by the tyrosine phosphorylation of fyn in Nb2 cells.

scholarly journals The v-Src SH3 domain binds phosphatidylinositol 3'-kinase.

1993 ◽  
Vol 13 (9) ◽  
pp. 5225-5232 ◽  
Author(s):  
X Liu ◽  
L E Marengere ◽  
C A Koch ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Chicken Embryo ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Src Tyrosine Kinase ◽  
Amino Terminal ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.

Inactivation of c-Yes tyrosine kinase by elevation of intracellular calcium levels

1993 ◽  
Vol 13 (12) ◽  
pp. 7507-7514
Author(s):  
Y Zhao ◽  
H Uyttendaele ◽  
J G Krueger ◽  
M Sudol ◽  
H Hanafusa
Keyword(s):  
Tyrosine Kinase ◽  
Intracellular Calcium ◽  
Protein Tyrosine Phosphatases ◽  
Cell Extract ◽  
Cellular Proteins ◽  
Src Tyrosine Kinase ◽  
Protein Levels ◽  
Kinase C ◽  
Dependent Inactivation

We have previously shown that the c-Src tyrosine kinase is activated four- to fivefold when cultured keratinocytes differentiate following the elevation of intracellular calcium levels. In contrast to c-Src, another Src family tyrosine kinase, c-Yes, was rapidly inactivated in these same cells, despite its marked similarity in structure and enzymatic activity to c-Src. The inactivation of c-Yes was independent of the protein kinase C pathway, which is usually activated by elevation of intracellular calcium levels. The protein levels of c-Src and c-Yes were not altered, but the phosphotyrosine content of both proteins was greatly reduced. As has been demonstrated for c-Src, in vitro dephosphorylation of c-Yes by incubation with protein tyrosine phosphatases also resulted in its activation, not inactivation. In vitro reconstitution experiments showed that c-Yes can be inactivated by preincubation with a Ca(2+)-supplemented cell extract and that this inhibition was reversed by the addition of EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. Gradient sedimentation of cell lysates showed that in cells treated with calcium and ionophore, c-Yes formed complexes with two distinct cellular proteins, whereas similar complexes were not seen in c-Src immunoprecipitates. One of these two proteins has the ability to inhibit c-Yes kinase activity in vitro. Finally, the Ca(2+)-dependent inactivation of c-Yes was observed in kidney tubular cells and fibroblasts, suggesting that the Ca(2+)-dependent regulation of c-Yes tyrosine kinase is not unique to keratinocytes. We postulate that c-Yes is inactivated through a Ca2+ -dependent association with cellular proteins, which seems to override its activation resulting from tyrosine dephosphorylation.

p60src and p72Syk Tyrosine Kinases Activate Both Lw and CD44 to Mediate Sickle Red Blood Cell Adhesion to Endothelium

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3223-3223
Author(s):  
Edward Chiou ◽  
Rahima Zennadi
Keyword(s):  
Shear Stress ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Conflicts Of Interest ◽  
Selective Inhibitor ◽  
Intermittent Flow ◽  
Αvβ3 Integrin ◽  
Src Tyrosine Kinase

Abstract Abstract 3223 The vaso-occlusive process in patients with sickle cell disease is complex and involves interactions between hemoglobin S red blood cells (SSRBCs) and vascular endothelial cells (ECs). However, the pathophysiologic triggers inducing SSRBC adhesion and vaso-occlusion are poorly understood. Elucidation of these mechanisms at the molecular level would allow development of new preventive and treatment strategies to abrogate vaso-occlusive events. Because non-receptor tyrosine kinases in non-erythroid cells are known to mediate cell-cell interactions, we have now investigated the role of non-receptor tyrosine kinases in modulating SSRBC adherence to cultured ECs, identified the kinases involved, and defined both the receptors on activated SSRBCs and the ligands on ECs involved in these interactions. Less than 20% of non-treated SSRBCs were able to adhere to non-stimulated ECs in vitro in intermittent flow conditions at a shear stress of 2 dynes/cm2. However, treatment of SSRBCs with sodium orthovanadate (Na3VO4), a potent broad spectrum inhibitor of protein tyrosine phosphatases, was able to significantly up-regulate RBC adhesion to cultured ECs by 4.6±1-fold over baseline adhesion in vitro. Na3VO4 in contrast, completely failed to increase adhesion of normal RBCs to cultured ECs. The increased SSRBC adhesion induced by Na3VO4 was significantly inhibited with piceatannol (p<0.001), which inhibits non-receptor tyrosine kinases p72Syk and p56Lck, and PP1 (p<0.001), an src-selective tyrosine p56Lck, p59Fyn and p60src kinase inhibitor. However, genistein, a broad range inhibitor of tyrosine kinases, and damnacanthal, a highly potent and selective inhibitor of p56Lck, completely failed to inhibit the effect of Na3VO4 on SSRBC adhesion. In addition, phenylarsine oxide, which specifically activates p56Lck tyrosine kinase, did not affect SSRBC adhesion to ECs. Together, these data suggest that SSRBC adhesion to ECs can be upregulated via activation of at least p72Syk and p60src tyrosine kinases, but not via the src-tyrosine kinase p56Lck. We further confirmed that increased SSRBC adhesion by Na3VO4 treatment is indeed a result of the activation of p72Syk and p60src tyrosine kinases. Slight basal phosphorylation of p72Syk and p60src was detected in all SSRBC samples tested. However, Na3VO4-treatment of SSRBCs significantly enhanced phosphorylation of both p60src and p72Syk kinases over basal phosphorylation (p<0.05), and this effect induced by Na3VO4 was completely blocked with the src-selective inhibitor PP1 or PP2, and piceatannol, respectively, suggesting that p72Syk and p60src undergo enhanced activation and are involved in up-regulation of SSRBC adhesion to endothelium. Moreover, at a shear stress of 2 dynes/cm2, both anti-LW (ICAM-4) and anti-CD44 antibodies individually inhibited adhesion of activated SSRBCs to ECs, by up to 83% and 78%, respectively. Recombinant soluble LW (srLW) and CD44 (srCD44) proteins also completely abolished adhesion of activated SSRBCs to ECs, identifying LW and CD44 as the RBC receptors involved in this interaction. The EC ligands for activated SSRBCs were also identified using antibody inhibition studies, as the αvβ3 integrin, a ligand previously shown to be important to SSRBC adhesion to activated ECs in vivo, and the endothelial CD44. These data demonstrate that activation of p72Syk and p60src-dependent pathways can act to activate LW- and CD44-mediated SSRBC adhesion to endothelial αvβ3 integrin and CD44, respectively, suggesting that this mechanism may initiate or exacerbate vaso-occlusion by increasing SSRBC adhesion to the endothelium. RBC CD44 is also the first adhesion molecule shown to be involved in SSRBC adhesion to endothelium. Disclosures: No relevant conflicts of interest to declare.

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