scholarly journals Phosphorylation of p27Kip1 Regulates Assembly and Activation of Cyclin D1-Cdk4

2008 ◽  
Vol 28 (20) ◽  
pp. 6462-6472 ◽  
Author(s):  
Michelle D. Larrea ◽  
Jiyong Liang ◽  
Thiago Da Silva ◽  
Feng Hong ◽  
Shan H. Shao ◽  
...  
Keyword(s):  
Complex Formation ◽  
Cyclin D1 ◽  
Tyrosine Phosphorylation ◽  
Combined Therapy ◽  
Src Family Kinases ◽  
Kinase Activation ◽  
Constitutive Activation ◽  
Catalytic Activation ◽  
Inhibitory Function

ABSTRACT p27 mediates Cdk2 inhibition and is also found in cyclin D1-Cdk4 complexes. The present data support a role for p27 in the assembly of D-type cyclin-Cdk complexes and indicate that both cyclin D1-Cdk4-p27 assembly and kinase activation are regulated by p27 phosphorylation. Prior work showed that p27 can be phosphorylated by protein kinase B/Akt (PKB/Akt) at T157 and T198. Here we show that PKB activation and the appearance of p27pT157 and p27pT198 precede p27-cyclin D1-Cdk4 assembly in early G1. PI3K/PKB inhibition rapidly reduced p27pT157 and p27pT198 and dissociated cellular p27-cyclin D1-Cdk4. Mutant p27 allele products lacking phosphorylation at T157 and T198 bound poorly to cellular cyclin D1 and Cdk4. Cellular p27pT157 and p27pT198 coprecipitated with Cdk4 but were not detected in Cdk2 complexes. The addition of p27 to recombinant cyclin D1 and Cdk4 led to cyclin D1-Cdk4-p27 complex formation in vitro. p27 phosphorylation by PKB increased p27-cyclin D1-Cdk4 assembly in vitro but yielded inactive Cdk4. In contrast, Src pretreatment of p27 did not affect p27-cyclin D1-Cdk4 complex formation. However, Src treatment led to tyrosine phosphorylation of p27 and catalytic activation of assembled cyclin D1-Cdk4-p27 complexes. Thus, while PKB-dependent p27 phosphorylation appears to increase cyclin D1-Cdk4-p27 assembly or stabilize these complexes in vitro, cyclin D1-Cdk4-p27 activation requires the tyrosine phosphorylation of p27. Constitutive activation of PKB and Abl or Src family kinases in cancers would drive p27 phosphorylation, increase cyclin D1-Cdk4 assembly and activation, and reduce the cyclin E-Cdk2 inhibitory function of p27. Combined therapy with both Src and PI3K/PKB inhibitors may reverse this process.

scholarly journals Regulation of 3-Phosphoinositide-dependent Protein Kinase-1 (PDK1) by Src Involves Tyrosine Phosphorylation of PDK1 and Src Homology 2 Domain Binding

2007 ◽  
Vol 283 (3) ◽  
pp. 1480-1491 ◽  
Author(s):  
Keum-Jin Yang ◽  
Sanghee Shin ◽  
Longzhen Piao ◽  
Eulsoon Shin ◽  
Yuwen Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Complex Formation ◽  
Tyrosine Phosphorylation ◽  
Immunohistochemical Analysis ◽  
Sh2 Domain ◽  
Resting Cells ◽  
Dependent Protein Kinase ◽  
Hek 293 ◽  
Dependent Protein

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr9 and Tyr373/376) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr9 antibodies showed that the level of Tyr9 phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr9, distinct from Tyr373/376, is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr9 phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.

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 Differential Activation of the Tyrosine Kinases ZAP-70 and Syk After FcγRI Stimulation

Blood ◽  
1997 ◽  
Vol 89 (2) ◽  
pp. 388-396 ◽  
Author(s):  
Naomi Taylor ◽  
Thomas Jahn ◽  
Susan Smith ◽  
Thomas Lamkin ◽  
Lisa Uribe ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Signal Transduction Pathway ◽  
Protein Tyrosine Kinases ◽  
Monocytic Cell ◽  
Monocytic Cells ◽  
Kinase Activation ◽  
Receptor Aggregation

Abstract Engagement of the high-affinity IgG Fc receptor (FcγRI) activates a signal transduction pathway involving tyrosine phosphorylation of associated kinases. We compared the activation of the related protein tyrosine kinases (PTKs), Syk and ZAP-70, in FcγRI-mediated signaling. Cross-linking of the FcγRI multimeric receptor in monocytic cells results in tyrosine phosphorylation of the FcεRIγ subunit and association of Syk with this complex. We stably introduced ZAP-70 via a retroviral vector into two monocytic cell lines, U937 and THP-1, which normally do not express ZAP-70. Neither Syk nor MAP kinase activation was affected by the presence of ZAP-70. Although transduced ZAP-70 had in vitro kinase activity and associated with FcεRIγ after receptor aggregation, it was not tyrosine phosphorylated. In contrast, both ZAP-70 and Syk were phosphorylated in a T-cell line in which their respective levels of expression were similar to those detected in U937/ZAP-70 cells. Therefore, these results suggest that requirements for Syk and ZAP-70 phosphorylation are distinct in a monocytic cell context.

scholarly journals SRC family kinases in hamster spermatozoa: evidence for the presence of LCK

Reproduction ◽  
2017 ◽  
Vol 153 (5) ◽  
pp. 655-669 ◽  
Author(s):  
Durgesh Kumar Singh ◽  
Rohit Kumar Deshmukh ◽  
Praveen Kumar Narayanan ◽  
Sisinthy Shivaji ◽  
Archana Bharadwaj Siva
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Specific Inhibitor ◽  
Specific Protein ◽  
Src Family Kinases ◽  
Sperm Capacitation ◽  
Protein Tyrosine

Sperm capacitation is a prerequisite for successful fertilization. Increase in tyrosine phosphorylation is considered the hallmark of capacitation and attempts to understand its regulation are ongoing. In this regard, we attempted to study the role of SRC family kinases (SFKs) in the hamster sperm functions. Interestingly, we found the presence of the lymphocyte-specific protein tyrosine kinase, LCK, in mammalian spermatozoa and further characterized it in terms of its localization and function. LCK was found in spermatozoa of several species, and its transcript was identified in the hamster testis. Autophosphorylation of LCK at the Y394 residue increased as capacitation progressed, indicating an upregulation of LCK activity during capacitation. Inhibition of LCK (and perhaps the other SFKs) with the use of a specific inhibitor showed a significant decrease in protein tyrosine phosphorylation of several proteins, implying LCK/SFKs as key tyrosine kinase(s) regulating tyrosine phosphorylation during hamster sperm capacitation. Dihydrolipoamide dehydrogenase was identified as a substrate for LCK/SFK. LCK/SFKs inhibition significantly reduced the percentage fertilization (in vitro) but had no effect on sperm motility, hyperactivation and acrosome reaction. In summary, this is the first report on the presence of LCK, an SFK of hematopoietic lineage in spermatozoa besides being the first study on the role of SFKs in the spermatozoa of Syrian hamsters.

scholarly journals Epithelial Cell Adhesion to Extracellular Matrix Proteins Induces Tyrosine Phosphorylation of the Epstein-Barr Virus Latent Membrane Protein 2: a Role for C-Terminal Src Kinase

1999 ◽  
Vol 73 (6) ◽  
pp. 4767-4775 ◽  
Author(s):  
Frank Scholle ◽  
Richard Longnecker ◽  
Nancy Raab-Traub
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Tyrosine Phosphorylation ◽  
Epstein Barr Virus ◽  
Latent Membrane Protein ◽  
Src Family Kinases ◽  
Barr Virus ◽  
Epstein Barr ◽  
Latent Membrane Protein 2

ABSTRACT The Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) is expressed in latently EBV-infected B cells, where it forms patches in the plasma membrane and interferes with B-cell receptor signal transduction through dominant-negative effects on protein kinases. LMP2 transcripts are detected in nasopharyngeal carcinoma, an epithelial-cell malignancy. In this study the function of LMP2A in epithelial cells was investigated. LMP2A was found to coprecipitate with protein kinase activities and to become phosphorylated in in vitro kinase assays. Analysis of LMP2A deletion mutants demonstrated that tyrosines implicated in interacting with Src family kinase SH2 domains and the SH2 domain of Csk, as well as the LMP2A immunoreceptor tyrosine-based activation motif, are important for its phosphorylation in epithelial cells. LMP2A tyrosine phosphorylation was triggered by cell adhesion to extracellular-matrix (ECM) proteins. Src family kinases, whose involvement in cell-ECM signaling and LMP2A phosphorylation in B lymphocytes has been well established, were found not to be responsible for LMP2A phosphorylation in epithelial cells. Instead, coexpression of Csk, a negative Src regulator, and LMP2A led to an increase in LMP2A phosphorylation both in nonadherent cells and upon cell adhesion. Csk also phosphorylated LMP2A in vitro. These results suggest that LMP2A has a different role in epithelial cells, where it interacts with cell adhesion-initiated signaling pathways. Although tyrosine phosphorylation of LMP2A occurs in both cell types, different protein kinases seem to be used: Src family kinases in B lymphocytes and Csk in epithelial cells.

scholarly journals Inhibition of Src Family Kinases by a Combinatorial Action of 5′-AMP and Small Heat Shock Proteins, Identified from the Adult Heart

1999 ◽  
Vol 19 (10) ◽  
pp. 6858-6871 ◽  
Author(s):  
Vijaykumar S. Kasi ◽  
Dhandapani Kuppuswamy
Keyword(s):  
Heat Shock ◽  
Tyrosine Phosphorylation ◽  
Cellular Proliferation ◽  
Catalytic Domain ◽  
Direct Interaction ◽  
Small Heat Shock Protein ◽  
Peptide Sequencing ◽  
Src Family Kinases

ABSTRACT Src family kinases are implicated in cellular proliferation and transformation. Terminally differentiated myocytes have lost the ability to proliferate, indicating the existence of a down-regulatory mechanism(s) for these mitogenic kinases. Here we show that feline cardiomyocyte lysate contains thermostable components that inhibit c-Src kinase in vitro. This inhibitory activity, present predominantly in heart tissue, involves two components acting combinatorially. After purification by sequential chromatography, one component was identified by mass and nuclear magnetic resonance spectroscopies as 5′-AMP, while the other was identified by peptide sequencing as a small heat shock protein (sHSP). 5′-AMP and to a lesser extent 5′-ADP inhibit c-Src when combined with either HSP-27 or HSP-32. Other HSPs, including αB-crystallin, HSP-70, and HSP-90, did not exhibit this effect. The inhibition, observed preferentially on Src family kinases and independent of the Src tyrosine phosphorylation state, occurs via a direct interaction of the c-Src catalytic domain with the inhibitory components. Our study indicates that sHSPs increase the affinity of 5′-AMP for the c-Src ATP binding site, thereby facilitating the inhibition. In vivo, elevation of ATP levels in the cardiomyocytes results in the tyrosine phosphorylation of cellular proteins including c-Src at the activatory site, and this effect is blocked when the 5′-AMP concentration is raised. Thus, this study reveals a novel role for sHSPs and 5′-AMP in the regulation of Src family kinases, presumably for the maintenance of the terminally differentiated state.

scholarly journals BCR-ABL and Src Family Kinases Induce YAP Tyrosine Phosphorylation Resulting in Survivin and Cyclin D1 Expression in Chronic Myeloid Leukemia Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5366-5366
Author(s):  
Toshiyuki Hori ◽  
Kenta Moriyama
Keyword(s):  
Cell Growth ◽  
Cyclin D1 ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Hippo Pathway ◽  
Src Family Kinases ◽  
Growth And Survival ◽  
Downstream Signaling ◽  
Erythroleukemia Cell

Chronic myeloid leukemia (CML) is caused by the BCR-ABL fusion protein. To date, several downstream signaling pathways of BCR-ABL have been reported to underlie the leukemogenesis of CML such as the JAK/STAT pathway, the PI3K/AKT pathway, and the Grb2/MAPK pathway. Furthermore, the Src family kinases (SFKs), especially Hck, Lyn and Fyn, have been suggested to be involved in BCR-ABL-induced transformation. Although these studies have revealed important aspects of the downstream signals of BCR-ABL, the detailed molecular mechanism of CML has not been thoroughly elucidated. Yes-associated protein (YAP) is a transcriptional cofactor that functions as an effector of the Hippo pathway which regulates cell growth and survival. In the classical Hippo pathway, YAP phosphorylated at serine 127 (S127) by LATS1/2 is bound to 14-3-3 and prevented from nuclear translocation. Apart from this serine/threonine phosphorylation, YAP undergoes phosphorylation at several tyrosine residues by various kinases to be activated. SFKs can phosphorylate and activate YAP, which has been demonstrated in some tumors. Among the several possible phosphorylated tyrosine residues, the phosphorylation at Y357 (p-Y357) has been demonstrated to be the most important for tumorigenesis. Therefore, it is possible that BCR-ABL directly or indirectly phosphorylates YAP through SFKs and thus activated YAP is translocated into the nucleus and together with TEAD induces expression of genes necessary for cell growth and survival. In the present study, we investigated the effects of imatinib and an SFK-specific inhibitor RK-20449 on viable cell number, YAP p-Y357 and expression of Survivin as well as Cyclin D1 in CML-derived cell lines in comparison with AML-derived cell lines. Furthermore, we established BCR-ABL stable transfectants and the control lines derived from TF-1, a factor dependent human erythroleukemia cell line, in order to verify our results obtained with CML-derived cell lines. We first checked the phosphorylation status of YAP and found that it was constitutively phosphorylated at tyrosine 357in CML-derived cell lines (TCC-S and K562) but not in AML-derived cell lines (HL-60 and KG-1a). Treatment with imatinib or RK-20449 inhibited cell growth and decreased tyrosine phosphorylation of YAP in both CML lines. Expression of Survivin or Cyclin D1 was decreased at least in TCC-S but not in AML cell lines. Furthermore, we established BCR-ABL stable transfectant and control empty vector transfectant from TF-1, a factor dependent human erythroleukemia cell line, in order to verify our results obtained with CML cell lines. YAP was phosphorylated at Y357 constitutively in BCR-ABL stable transfectant but not in control transfectant, and treatment with imatinib or RK-20449 inhibited cell growth, YAP tyrosine phosphorylation, and expression of Cyclin D1 in BCR-ABL stable transfectant. These results suggest that BCR-ABL induces tyrosine phosphorylation of YAP presumably through Src family kinases, which results in expression of Survivin and Cyclin D leading to leukemogenesis in CML cells. We have not determined which SFK is involved in the downstream signaling of BCR-ABL. Overexpression experiments in HEK293T have indicated that Lck, Lyn and Fyn can phosphorylate YAP at Y357 without BCR-ABL. However, it remains to be determined which SFK is activated by BCR-ABL and practically involved in YAP phosphorylation resulting in leukemogenesis. Further studies are required to specify the relevant SFKs and disclose the downstream signaling from activated YAP. Disclosures No relevant conflicts of interest to declare.

The focal adhesion kinase Pyk2 links Ca2+ signalling to Src family kinase activation and protein tyrosine phosphorylation in thrombin-stimulated platelets

2015 ◽  
Vol 469 (2) ◽  
pp. 199-210 ◽  
Author(s):  
Ilaria Canobbio ◽  
Lina Cipolla ◽  
Gianni F. Guidetti ◽  
Daria Manganaro ◽  
Caterina Visconte ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Blood Platelets ◽  
Src Family Kinases ◽  
G Protein Coupled Receptors ◽  
Protein Tyrosine Phosphorylation ◽  
Kinase Activation ◽  
Protein Tyrosine ◽  
Dependent Protein ◽  
G Protein Coupled

We address the mechanism for Src family kinases activation downstream of G-protein-coupled receptors (GPCRs) in thrombin-stimulated blood platelets and we describe a novel interplay between Pyk2 and the Src kinases Fyn and Lyn in the regulation of Ca2+-dependent protein-tyrosine phosphorylation.

scholarly journals Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis

2011 ◽  
Vol 193 (1) ◽  
pp. 185-199 ◽  
Author(s):  
Jenna L. Jewell ◽  
Eunjin Oh ◽  
Latha Ramalingam ◽  
Michael A. Kalwat ◽  
Vincent S. Tagliabracci ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Phosphatidylinositol 3 Kinase ◽  
Core Complex ◽  
Kinase Activation ◽  
Vesicle Exocytosis ◽  
Syntaxin 4 ◽  
Acid Reduction

How the Sec1/Munc18–syntaxin complex might transition to form the SNARE core complex remains unclear. Toward this, Munc18c tyrosine phosphorylation has been correlated with its dissociation from syntaxin 4. Using 3T3-L1 adipocytes subjected to small interfering ribonucleic acid reduction of Munc18c as a model of impaired insulin-stimulated GLUT4 vesicle exocytosis, we found that coordinate expression of Munc18c–wild type or select phosphomimetic Munc18c mutants, but not phosphodefective mutants, restored GLUT4 vesicle exocytosis, suggesting a requirement for Munc18c tyrosine phosphorylation at Tyr219 and Tyr521. Surprisingly, the insulin receptor (IR) tyrosine kinase was found to target Munc18c at Tyr521 in vitro, rapidly binding and phosphorylating endogenous Munc18c within adipocytes and skeletal muscle. IR, but not phosphatidylinositol 3-kinase, activation was required. Altogether, we identify IR as the first known tyrosine kinase for Munc18c as part of a new insulin-signaling step in GLUT4 vesicle exocytosis, exemplifying a new model for the coordination of SNARE assembly and vesicle mobilization events in response to a single extracellular stimulus.

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