Src Family Of Protein Kinases Research Articles

Protein Palmitoylation in Leukocyte Signaling and Function.

Palmitoylation is a post-translational modification (PTM) based on thioester-linkage between palmitic acid and the cysteine residue of a protein. This covalent attachment of palmitate is reversibly and dynamically regulated by two opposing sets of enzymes: palmitoyl acyltransferases containing a zinc finger aspartate-histidine-histidine-cysteine motif (PAT-DHHCs) and thioesterases. The reversible nature of palmitoylation enables fine-tuned regulation of protein conformation, stability, and ability to interact with other proteins. More importantly, the proper function of many surface receptors and signaling proteins requires palmitoylation-meditated partitioning into lipid rafts. A growing number of leukocyte proteins have been reported to undergo palmitoylation, including cytokine/chemokine receptors, adhesion molecules, pattern recognition receptors, scavenger receptors, T cell co-receptors, transmembrane adaptor proteins, and signaling effectors including the Src family of protein kinases. This review provides the latest findings of palmitoylated proteins in leukocytes and focuses on the functional impact of palmitoylation in leukocyte function related to adhesion, transmigration, chemotaxis, phagocytosis, pathogen recognition, signaling activation, cytotoxicity, and cytokine production.

Abstract C191: Preclinical investigation of the antitumor efficacy in lung cancer of Y15, a novel focal adhesion kinase inhibitor.

Abstract Introduction: Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that associates with a number of cell adhesion components, such as integrins, paxillin and talin, as well as with various signaling proteins such as phosphatidylinositol 3-kinase (PI3K) and the Src family of protein kinases. It thus plays an important role in cell motility, invasion, growth and cell survival. Y15 (1,2,4,5-benzentetraamine tetra hydrochloride) is a small molecule that inhibits FAK autophosphorylation by selectively targeting the Y397 phosphorylation site of FAK. It has shown activity against breast, colon and pancreatic cancers. We herein present data in lung cancer. Methods: Various lung cancer cell lines as well as minimally passaged patient-derived tumor (containing PIK3CA mutation E545K) propagated in vitro were treated with escalating doses of Y15 or DMSO for 72 hours to evaluate single-agent activity. Cell lines were also treated with cytotoxic agents (cisplatin, gemcitabine, pemetrexed, paclitaxel) in combination with escalating doses of Y15. Cellular viability was assessed by trypan blue exclusion assay and colony formation assay. For in vivo experiments, selected cell lines were implanted into nude mice and vehicle versus Y15 treatment, via either intraperitoneal or oral gavage administration, once daily was started when tumors reached a mean volume of 50–60 mm3. Western blot will be performed to investigate alterations in the activity of various signaling proteins. Results: Y15 exhibited time and dose-dependent cell death in 6 out of 9 lung cancer cell lines tested. Activity was demonstrated beginning at 4 uM dose, with almost 100% cell kill demonstrated at 10 uM dose. There was also corresponding dose-dependent loss of clonogenic ability starting at 1 uM dose in sensitive cell lines. In addition, Y15 enhanced paclitaxel-induced cell death. Mice treated with Y15 at 100 mg/kg oral gavage administration showed reduced tumor growth compared to control mice. Conclusion: Y15 demonstrates promising antitumor efficacy against various non-small cell lung cancer cell lines. Further development of this agent in lung cancer is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C191.

Abstract 2936: MicroRNA-205 interdiction of Src-mediated oncogenic pathways in renal cancer

Abstract Introduction and Objective: The Src family of protein kinases (SFKs) plays key roles in regulating fundamental cellular processes, including cell growth, differentiation, cell shape, migration and survival, and specialized cell signals in various malignancies. The pleotropic functions of SFKs in cancer make them promising targets for intervention. We investigated the role of miR-205 in inhibition of Src-mediated oncogenic pathways in renal cancer. Methods: The methods employed in this study include quantitative-real time PCR; western blot; fluorescence-activated cell sorting assays for cell cycle distribution and apoptosis; assays for cell viabiltity, clonability, migration and invasiveness of prostate cancer cells. Luciferase reporter and mutational assays; generation of miR-205 stable cells and in-vivo study in nude mice was also performed. Results: The expression of miR-205 was significantly suppressed in renal cancer cell lines and tumors when compared with normal tissues and a non-malignant cell line, and is correlated inversely with the expression of SFKs. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3’UTR sequences complementary to either Src, Lyn or Yes, which was abolished by mutations in these 3’UTR regions. Over-expression of miR-205 in A498 cells reduced Src, Lyn and Yes expression both at mRNA and protein levels. Proliferation of renal cancer cells was suppressed by miR-205, mediated by the phosphoSrc-regulated ERK1/2 pathway. Cell motility factor- FAK and STAT3 activation was also inhibited by miR-205. Transient as well as stable over-expression of miR-205 in A498 cells resulted in induction of G0/G1 cell cycle arrest and apoptosis as indicated by decreased levels of cyclin D1 and cMyc, suppressed cell proliferation, colony formation, migration, and invasion in renal cancer cells. miR-205 also inhibited tumor cell growth in vivo. This is the first study demonstrating that miRNA-205 inhibits proto-oncogenic Src family of kinases indicating a therapeutic potential of miR-205 in the treatment of renal cancer. Conclusion: This study demonstrates that miRNA-205 inhibits proto-oncogenic Src family of kinases indicating a therapeutic potential of miR-205 in the treatment of renal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2936. doi:10.1158/1538-7445.AM2011-2936

MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

The Src family of protein kinases (SFK) plays key roles in regulating fundamental cellular processes, including cell growth, differentiation, cell shape, migration, and survival, and specialized cell signals in various malignancies. The pleiotropic functions of SFKs in cancer make them promising targets for intervention. Here, we sought to investigate the role of microRNA-205 (miR-205) in inhibition of Src-mediated oncogenic pathways in renal cancer. We report that expression of miR-205 was significantly suppressed in renal cancer cell lines and tumors when compared with normal tissues and a nonmalignant cell line and is correlated inversely with the expression of SFKs. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR (untranslated region) sequences complementary to either Src, Lyn, or Yes, which was abolished by mutations in these 3'-UTR regions. Overexpression of miR-205 in A498 cells reduced Src, Lyn, and Yes expression, both at mRNA and protein levels. Proliferation of renal cancer cells was suppressed by miR-205, mediated by the phospho-Src-regulated ERK1/2 pathway. Cell motility factor FAK (focal adhesion kinase) and STAT3 activation were also inhibited by miR-205. Transient and stable overexpression of miR-205 in A498 cells resulted in induction of G₀/G₁ cell-cycle arrest and apoptosis, as indicated by decreased levels of cyclin D1 and c-Myc, suppressed cell proliferation, colony formation, migration, and invasion in renal cancer cells. miR-205 also inhibited tumor cell growth in vivo. This is the first study showing that miR-205 inhibits proto-oncogenic SFKs, indicating a therapeutic potential of miR-205 in the treatment of renal cancer.

Signaling and regulation of the platelet glycoprotein Ib–IX–V complex

The platelet adhesion receptor, the glycoprotein Ib-IX-V complex, not only mediates platelet adhesion but also transmits signals leading to platelet activation, aggregation and secretion. Significant progress has been made recently on the signaling pathways and regulatory mechanisms involving glycoprotein Ib-IX-V function. The interaction of glycoprotein Ib-IX-V with its ligand, von Willebrand factor, is dually controlled by von Willebrand factor conformation and intracellular signal-mediated regulation of glycoprotein Ib-IX-V receptor function that requires the zeta isoform of the 14-3-3 protein family (14-3-3zeta). Glycoprotein Ib-IX-V signaling is mediated by the Src family of protein kinases, phospholipase C, calcium elevation, phosphoinositol 3-kinase, and multiple amplification mechanisms including the nitric oxide-cGMP pathway, the mitogen-activated protein kinase pathway, the immunoreceptor tyrosine-based activation motif pathway, and ADP and thromboxane A2 pathways. Progress in understanding the mechanism(s) regulating glycoprotein Ib-IX-V should help develop inhibitors and modifiers that interfere or augment its von Willebrand factor binding function and thus be useful for treating thrombosis and bleeding disorders. Characterization of intracellular molecules and pathways in glycoprotein Ib-IX-V signaling has implications in the development of new agents and for the use of existing drugs that affect glycoprotein Ib-IX-V signaling.

C-terminal Src Kinase-homologous Kinase (CHK), a Unique Inhibitor Inactivating Multiple Active Conformations of Src Family Tyrosine Kinases

The Src family of protein kinases (SFKs) mediates mitogenic signal transduction, and constitutive SFK activation is associated with tumorigenesis. To prevent constitutive SFK activation, the catalytic activity of SFKs in normal mammalian cells is suppressed mainly by two inhibitors called C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK), which inactivate SFKs by phosphorylating a consensus tyrosine near the C terminus of SFKs (Y(T)). The phosphorylated Y(T) intramolecularly binds to the SH2 domain of SFKs. This interaction, known as pY(T)/SH2 interaction, together with binding between the SH2 kinase linker and the SH3 domain of SFKs (linker/SH3 interaction) stabilizes SFKs in a "closed" inactive conformation. We previously discovered an alternative mechanism CHK employs to inhibit SFKs. This mechanism, referred to as the non-catalytic inhibitory mechanism, involves tight binding of CHK to SFKs; the binding alone is sufficient to inhibit SFKs. Herein, we constructed multiple active conformations of an SFK member, Hck, by systematically disrupting the two inhibitory interactions. We found that CHK employs the non-catalytic mechanism to inactivate these active conformations of Hck. However, CHK does not bind Hck when it adopts the inactive conformation in which both inhibitory interactions are intact. These data indicate that binding of CHK to SFKs via the non-catalytic mechanism is governed by the conformations of SFKs. Although CSK is also an inhibitor of SFKs, it does not inhibit SFKs by a similar non-catalytic mechanism. Thus, the non-catalytic inhibitory mechanism is a unique property of CHK that allows it to down-regulate multiple active conformations of SFKs.

Tyrosine Phosphorylation of the Integrin β3 Subunit Regulates β3 Cleavage by Calpain

Outside-in signaling of beta(3) integrins induces and requires phosphorylation at tyrosine 747 (Tyr(747)) and tyrosine 759 (Tyr(759)) of the beta(3) subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of beta(3) cytoplasmic domain. Here we show that beta(3) tyrosine phosphorylation inhibits calpain cleavage. Mutating both tyrosines to phenylalanine sensitizes beta(3) to calpain cleavage. Furthermore, phosphorylation at Tyr(747) and Tyr(759) of beta(3) in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Tyr(759) is associated with calpain cleavage at Tyr(759). Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the beta(3) cytoplasmic domain.

Herpes Simplex Virus 1 Precludes Replenishment of the Short-Lived Receptor of Tumor Necrosis Factor Alpha by Virion Host Shutoff-Dependent Degradation of Its mRNA

Cell function is tightly regulated by surface receptors. Earlier reports showed that herpes simplex virus 1 regulates by diverse mechanisms the presentation of antigenic peptides, downregulates the signaling pathways associated with receptor tyrosine kinases, and posttranslationally modifies members of the Src family of protein kinases. Here we report that the receptor for tumor necrosis factor alpha (TNF-R1) rapidly disappears from both the cell surface and total cell lysates in cells infected with wild-type virus or a variety of mutants but not in cells infected with the mutant DeltaU(L)41, which lacks the U(L)41 gene, the virion host shutoff gene. The half-life of TNF-R1 appears to be less than 30 min in both mock-infected and infected cells. The disappearance of TNF-R1 correlates with the disappearance of cytoplasmic TNF-R1 mRNA in wild-type-virus-infected cells. The results suggest that by degrading the TNFR1 mRNA, the virus precludes the replenishment of naturally decaying TNF-R1.

The Molecular Perspective: c-Abl Tyrosine Kinase

The Oncologist 2005;10:758–759 www.TheOncologist.com Correspondence: David S. Goodsell, Ph.D., Associate Professor, The Scripps Research Institute, Department of Molecular Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Telephone: 858-784-2839; Fax: 858-784-2860; e-mail: goodsell@scripps.edu; Website: http://www.scripps.edu/pub/goodsell Received August 18, 2005; accepted for publication August 18, 2005. ©AlphaMed Press 10837159/2005/$12.00/0 Phosphate groups are widely used for transmitting signals inside cells. Phosphate has a number of advantages in molecular signaling. With its strong charge and numerous opportunities for hydrogen bonding, it is particularly easy to recognize. Phosphate groups are also readily added to and removed from signaling molecules, using the cell’s ready supply of ATP to power the process. Phosphate groups may be added to small molecules to create characteristic molecules like cyclic AMP. They may also be added to proteins, changing their surface features or even modulating the activity of an enzyme. As you can imagine, however, one phosphate group looks much like every other one. To be useful in signaling, the phosphate groups must be attached in the proper place and at the proper time. To perform this function, our cells have more than 500 different protein kinases, each designed to add phosphates to a different set of proteins. These many kinases are involved in a complex, interconnected network of signaling, requiring careful control so that each kinase is activated only when its particular signal is needed. The c-Abl tyrosine kinase, shown in Figure 1, transmits messages about the adhesion of cells to their neighbors and messages indicating when it is time to grow or move to a new location. Like other members of the src family of protein kinases, c-Abl uses a complex conformational change to turn its kinase activity on and off. It is comprised of several domains connected by flexible linkers, including two small regulatory domains, a larger kinase domain, and several additional domains that bind to DNA and actin. In the inactive form, the protein folds into Access and take the CME test online and receive 1 hour of AMA PRA category 1 credit at CME.TheOncologist.com CME The Molecular Perspective: c-Abl Tyrosine Kinase

Src-mediated Tyrosine Phosphorylation of Caveolin-1 Induces Its Association with Membrane Type 1 Matrix Metalloproteinase

We have recently shown that stimulation of endothelial cells with vascular endothelial growth factor (VEGF) induces dissociation of caveolin-1 from the VEGFR-2 receptor, followed by Src family kinase-dependent tyrosine phosphorylation of the protein (Labrecque, L., Royal, I., Surprenant, D. S., Patterson, C., Gingras, D., and Beliveau, R. (2003) Mol. Biol. Cell 14, 334-347). In this study, we provide evidence that the VEGF-dependent tyrosine phosphorylation of caveolin-1 induces interaction of the protein with the membrane-type 1 matrix metalloproteinase (MT1-MMP). This interaction requires the phosphorylation of caveolin-1 on tyrosine 14 by members of the Src family of protein kinases, such as Src and Fyn, because it is completely abolished by expression of a catalytically inactive Src mutant or by site-directed mutagenesis of tyrosine 14 of caveolin-1. Most interestingly, the association of MT1-MMP with phosphorylated caveolin-1 induced the recruitment of Src and a concomitant inhibition of the kinase activity of the enzyme, suggesting that this complex may be involved in the negative regulation of Src activity. The association of MT1-MMP with phosphorylated caveolin-1 occurs in caveolae membranes and involves the cytoplasmic domain of MT1-MMP because it was markedly reduced by mutation of Cys574 and Val582 residues of the cytoplasmic tail of the enzyme. Most interestingly, the reduction of the interaction between MT1-MMP and caveolin-1 by using these mutants also decreases MT1-MMP-dependent cell locomotion. Overall these results indicate that MT1-MMP associates with tyrosine-phosphorylated caveolin-1 and that this complex may play an important role in MT1-MMP regulation and function.

Analysis of styryl-based inhibitors of the lymphocyte tyrosine protein kinase p561ck

Several styryl-based compounds were evaluated for their capacity to act as inhibitors of the non-receptor tyrosine protein kinase p56lck. Our results demonstrate that alpha-cyanocinnamamide compounds can inhibit both the in vitro tyrosine autophosphorylation of p56lck as well as p56lck phosphorylation of exogenous substrates. Compound 67B-83-A was found to inhibit p56lck protein kinase activity with a calculated IC50 of 7 to 10 microM. This compound did not significantly inhibit the tyrosine protein kinase activity of the epidermal growth factor receptor and was found to be a less effective tyrosine protein kinase inhibitor for other members of the src family of protein kinases.