Browsing by Subject "MAP Kinase Signaling System"
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Item Open Access A Transcriptional Signature Identifies LKB1 Functional Status as a Novel Determinant of MEK Sensitivity in Lung Adenocarcinoma.(Cancer research, 2017-01) Kaufman, Jacob M; Yamada, Tadaaki; Park, Kyungho; Timmers, Cynthia D; Amann, Joseph M; Carbone, David PLKB1 is a commonly mutated tumor suppressor in non-small cell lung cancer that exerts complex effects on signal transduction and transcriptional regulation. To better understand the downstream impact of loss of functional LKB1, we developed a transcriptional fingerprint assay representing this phenotype. This assay was predictive of LKB1 functional loss in cell lines and clinical specimens, even those without detected sequence alterations in the gene. In silico screening of drug sensitivity data identified putative LKB1-selective drug candidates, revealing novel associations not apparent from analysis of LKB1 mutations alone. Among the candidates, MEK inhibitors showed robust association with signature expression in both training and testing datasets independent of RAS/RAF mutations. This susceptibility phenotype is directly altered by RNA interference-mediated LKB1 knockdown or by LKB1 re-expression into mutant cell lines and is readily observed in vivo using a xenograft model. MEK sensitivity is dependent on LKB1-induced changes in AKT and FOXO3 activation, consistent with genomic and proteomic analyses of LKB1-deficient lung adenocarcinomas. Our findings implicate the MEK pathway as a potential therapeutic target for LKB1-deficient cancers and define a practical NanoString biomarker to identify functional LKB1 loss. Cancer Res; 77(1); 153-63. ©2016 AACR.Item Open Access Across the meiotic divide - CSF activity in the post-Emi2/XErp1 era.(J Cell Sci, 2008-11-01) Wu, Judy Qiju; Kornbluth, SallyVertebrate eggs are arrested at the metaphase stage of meiosis II. Only upon fertilization will the metaphase-II-arrested eggs exit meiosis II and enter interphase. In 1971, Masui and Markert injected egg extracts into a two-cell-stage embryo and found that the injected blastomere arrested at the next mitosis. On the basis of these observations, they proposed the existence of an activity present in the eggs that is responsible for meiosis-II arrest and can induce mitotic arrest, and named this activity cytostatic factor (CSF). Although the existence of CSF was hypothesized more than 35 years ago, its precise identity remained unclear until recently. The discovery of the Mos-MAPK pathway and characterization of the anaphase-promoting complex/cyclosome (APC/C) as a central regulator of M-phase exit provided the framework for a molecular understanding of CSF. These pathways have now been linked by the discovery and characterization of the protein Emi2, a meiotic APC/C inhibitor, the activity and stability of which are controlled by the Mos-MAPK pathway. Continued investigation into the mechanism of action and mode of regulation of Emi2 promises to shed light not only on CSF function, but also on the general principles of APC/C regulation and the control of protein function by MAPK pathways.Item Open Access Arterial pole progenitors interpret opposing FGF/BMP signals to proliferate or differentiate.(Development, 2010-09) Hutson, MR; Zeng, XL; Kim, AJ; Antoon, E; Harward, S; Kirby, MLDuring heart development, a subpopulation of cells in the heart field maintains cardiac potential over several days of development and forms the myocardium and smooth muscle of the arterial pole. Using clonal and explant culture experiments, we show that these cells are a stem cell population that can differentiate into myocardium, smooth muscle and endothelial cells. The multipotent stem cells proliferate or differentiate into different cardiovascular cell fates through activation or inhibition of FGF and BMP signaling pathways. BMP promoted myocardial differentiation but not proliferation. FGF signaling promoted proliferation and induced smooth muscle differentiation, but inhibited myocardial differentiation. Blocking the Ras/Erk intracellular pathway promoted myocardial differentiation, while the PLCgamma and PI3K pathways regulated proliferation. In vivo, inhibition of both pathways resulted in predictable arterial pole defects. These studies suggest that myocardial differentiation of arterial pole progenitors requires BMP signaling combined with downregulation of the FGF/Ras/Erk pathway. The FGF pathway maintains the pool of proliferating stem cells and later promotes smooth muscle differentiation.Item Open Access Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.(Circ Res, 2008-07-03) Kim, Jihee; Zhang, Lisheng; Peppel, Karsten; Wu, Jiao-Hui; Zidar, David A; Brian, Leigh; DeWire, Scott M; Exum, Sabrina T; Lefkowitz, Robert J; Freedman, Neil JAtherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.Item Open Access Controlled and cardiac-restricted overexpression of the arginine vasopressin V1A receptor causes reversible left ventricular dysfunction through Gαq-mediated cell signaling.(Circulation, 2011-08) Li, Xue; Chan, Tung O; Myers, Valerie; Chowdhury, Ibrul; Zhang, Xue-Qian; Song, Jianliang; Zhang, Jin; Andrel, Jocelyn; Funakoshi, Hajime; Robbins, Jeffrey; Koch, Walter J; Hyslop, Terry; Cheung, Joseph Y; Feldman, Arthur MBackground
[Arg8]-vasopressin (AVP) activates 3 G-protein-coupled receptors: V1A, V2, and V1B. The AVP-V1A receptor is the primary AVP receptor in the heart; however, its role in cardiac homeostasis is controversial. To better understand AVP-mediated signaling in the heart, we created a transgenic mouse with controlled overexpression of the V1A receptor.Methods and results
The V1A receptor transgene was placed under the control of the tetracycline-regulated, cardiac-specific α-myosin heavy chain promoter (V1A-TG). V1A-TG mice had a normal cardiac function phenotype at 10 weeks of age; however, by 24 weeks of age, tetracycline-transactivating factor/V1A-TG mouse hearts had reduced cardiac function, cardiac hypertrophy, and dilatation of the ventricular cavity. Contractile dysfunction was also observed in isolated adult cardiac myocytes. When V1A receptor transgene was induced to be expressed in adult mice (V1A-TG(Ind)), left ventricular dysfunction and dilatation were also seen, albeit at a later time point. Because the V1A receptor mediates cell signaling through Gα(q) protein, we blocked Gα(q) signaling by crossing tetracycline-transactivating factor/V1A mice with transgenic mice that expressed a small inhibitory peptide against Gα(q). Gα(q) blockade abrogated the development of the heart failure phenotype in tetracycline-transactivating factor/V1A-TG mice. The heart failure phenotype could be reversed by administration of doxycycline.Conclusions
Our results demonstrate a role for V1A-mediated signaling in the development of heart failure and support a role for V1A blockade in the treatment of patients with elevated levels of vasopressin.Item Open Access CYLD inhibits melanoma growth and progression through suppression of the JNK/AP-1 and β1-integrin signaling pathways.(J Invest Dermatol, 2013-01) Ke, Hengning; Augustine, Christina K; Gandham, Vineela D; Jin, Jane Y; Tyler, Douglas S; Akiyama, Steven K; Hall, Russell P; Zhang, Jennifer YThe molecular mechanisms mediating cylindromatosis (CYLD) tumor suppressor function appear to be manifold. Here, we demonstrate that, in contrast to the increased levels of phosphorylated c-Jun NH(2)-terminal kinase (pJNK), CYLD was decreased in a majority of the melanoma cell lines and tissues examined. Exogenous expression of CYLD but not its catalytically deficient mutant markedly inhibited melanoma cell proliferation and migration in vitro and subcutaneous tumor growth in vivo. In addition, the melanoma cells expressing exogenous CYLD were unable to form pulmonary tumor nodules following tail-vein injection. At the molecular level, CYLD decreased β1-integrin and inhibited pJNK induction by tumor necrosis factor-α or cell attachment to collagen IV. Moreover, CYLD induced an array of other molecular changes associated with modulation of the "malignant" phenotype, including a decreased expression of cyclin D1, N-cadherin, and nuclear Bcl3, and an increased expression of p53 and E-cadherin. Most interestingly, coexpression of the constitutively active MKK7 or c-Jun mutants with CYLD prevented the above molecular changes, and fully restored melanoma growth and metastatic potential in vivo. Our findings demonstrate that the JNK/activator protein 1 signaling pathway underlies the melanoma growth and metastasis that are associated with CYLD loss of function. Thus, restoration of CYLD and inhibition of JNK and β1-integrin function represent potential therapeutic strategies for treatment of malignant melanoma.Item Open Access Deregulated PP1α phosphatase activity towards MAPK activation is antagonized by a tumor suppressive failsafe mechanism.(Nature communications, 2018-01-15) Chen, Ming; Wan, Lixin; Zhang, Jiangwen; Zhang, Jinfang; Mendez, Lourdes; Clohessy, John G; Berry, Kelsey; Victor, Joshua; Yin, Qing; Zhu, Yuan; Wei, Wenyi; Pandolfi, Pier PaoloThe mitogen-activated protein kinase (MAPK) pathway is frequently aberrantly activated in advanced cancers, including metastatic prostate cancer (CaP). However, activating mutations or gene rearrangements among MAPK signaling components, such as Ras and Raf, are not always observed in cancers with hyperactivated MAPK. The mechanisms underlying MAPK activation in these cancers remain largely elusive. Here we discover that genomic amplification of the PPP1CA gene is highly enriched in metastatic human CaP. We further identify an S6K/PP1α/B-Raf signaling pathway leading to activation of MAPK signaling that is antagonized by the PML tumor suppressor. Mechanistically, we find that PP1α acts as a B-Raf activating phosphatase and that PML suppresses MAPK activation by sequestering PP1α into PML nuclear bodies, hence repressing S6K-dependent PP1α phosphorylation, 14-3-3 binding and cytoplasmic accumulation. Our findings therefore reveal a PP1α/PML molecular network that is genetically altered in human cancer towards aberrant MAPK activation, with important therapeutic implications.Item Open Access Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells.(Cancer Biol Ther, 2010-01) Misra, Uma K; Pizzo, Salvatore VWe have previously shown that treatment of prostate cancer and melanoma cells expressing GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX and BAK expression as well as cleaved caspases-3, -7, -8 and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.Item Open Access STIM1-Ca(2+) signaling is required for the hypertrophic growth of skeletal muscle in mice.(Molecular and cellular biology, 2012-08) Li, Tianyu; Finch, Elizabeth A; Graham, Victoria; Zhang, Zhu-Shan; Ding, Jin-Dong; Burch, Jarrett; Oh-hora, Masatsugu; Rosenberg, PaulImmediately after birth, skeletal muscle must undergo an enormous period of growth and differentiation that is coordinated by several intertwined growth signaling pathways. How these pathways are integrated remains unclear but is likely to involve skeletal muscle contractile activity and calcium (Ca(2+)) signaling. Here, we show that Ca(2+) signaling governed by stromal interaction molecule 1 (STIM1) plays a central role in the integration of signaling and, therefore, muscle growth and differentiation. Conditional deletion of STIM1 from the skeletal muscle of mice (mSTIM1(-/-) mice) leads to profound growth delay, reduced myonuclear proliferation, and perinatal lethality. We show that muscle fibers of neonatal mSTIM1(-/-) mice cannot support the activity-dependent Ca(2+) transients evoked by tonic neurostimulation, even though excitation contraction coupling (ECC) remains unperturbed. In addition, disruption of tonic Ca(2+) signaling in muscle fibers attenuates downstream muscle growth signaling, such as that of calcineurin, mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1 and 2 (ERK1/2), and AKT. Based on our findings, we propose a model wherein STIM1-mediated store-operated calcium entry (SOCE) governs the Ca(2+) signaling required for cellular processes that are necessary for neonatal muscle growth and differentiation.Item Open Access The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration.(Cell, 2009-09-04) Yan, Dong; Wu, Zilu; Chisholm, Andrew D; Jin, YishiGrowth cone guidance and synaptic plasticity involve dynamic local changes in proteins at axons and dendrites. The Dual-Leucine zipper Kinase MAPKKK (DLK) has been previously implicated in synaptogenesis and axon outgrowth in C. elegans and other animals. Here we show that in C. elegans DLK-1 regulates not only proper synapse formation and axon morphology but also axon regeneration by influencing mRNA stability. DLK-1 kinase signals via a MAPKAP kinase, MAK-2, to stabilize the mRNA encoding CEBP-1, a bZip protein related to CCAAT/enhancer-binding proteins, via its 3'UTR. Inappropriate upregulation of cebp-1 in adult neurons disrupts synapses and axon morphology. CEBP-1 and the DLK-1 pathway are essential for axon regeneration after laser axotomy in adult neurons, and axotomy induces translation of CEBP-1 in axons. Our findings identify the DLK-1 pathway as a regulator of mRNA stability in synapse formation and maintenance and also in adult axon regeneration.Item Open Access The Host Response to Viral Infections Reveals Common and Virus-Specific Signatures in the Peripheral Blood.(Frontiers in immunology, 2021-01) Tsalik, Ephraim L; Fiorino, Cassandra; Aqeel, Ammara; Liu, Yiling; Henao, Ricardo; Ko, Emily R; Burke, Thomas W; Reller, Megan E; Bodinayake, Champica K; Nagahawatte, Ajith; Arachchi, Wasantha K; Devasiri, Vasantha; Kurukulasooriya, Ruvini; McClain, Micah T; Woods, Christopher W; Ginsburg, Geoffrey S; Tillekeratne, L Gayani; Schughart, KlausViruses cause a wide spectrum of clinical disease, the majority being acute respiratory infections (ARI). In most cases, ARI symptoms are similar for different viruses although severity can be variable. The objective of this study was to understand the shared and unique elements of the host transcriptional response to different viral pathogens. We identified 162 subjects in the US and Sri Lanka with infections due to influenza, enterovirus/rhinovirus, human metapneumovirus, dengue virus, cytomegalovirus, Epstein Barr Virus, or adenovirus. Our dataset allowed us to identify common pathways at the molecular level as well as virus-specific differences in the host immune response. Conserved elements of the host response to these viral infections highlighted the importance of interferon pathway activation. However, the magnitude of the responses varied between pathogens. We also identified virus-specific responses to influenza, enterovirus/rhinovirus, and dengue infections. Influenza-specific differentially expressed genes (DEG) revealed up-regulation of pathways related to viral defense and down-regulation of pathways related to T cell and neutrophil responses. Functional analysis of entero/rhinovirus-specific DEGs revealed up-regulation of pathways for neutrophil activation, negative regulation of immune response, and p38MAPK cascade and down-regulation of virus defenses and complement activation. Functional analysis of dengue-specific up-regulated DEGs showed enrichment of pathways for DNA replication and cell division whereas down-regulated DEGs were mainly associated with erythrocyte and myeloid cell homeostasis, reactive oxygen and peroxide metabolic processes. In conclusion, our study will contribute to a better understanding of molecular mechanisms to viral infections in humans and the identification of biomarkers to distinguish different types of viral infections.Item Open Access Tocopherol-associated protein suppresses prostate cancer cell growth by inhibition of the phosphoinositide 3-kinase pathway.(Cancer research, 2005-11) Ni, Jing; Wen, Xingqiao; Yao, Jorge; Chang, Hong-Chiang; Yin, Yi; Zhang, Min; Xie, Shaozhen; Chen, Ming; Simons, Brenna; Chang, Philip; di Sant'Agnese, Anthony; Messing, Edward M; Yeh, ShuyuanEpidemiologic studies suggested that vitamin E has a protective effect against prostate cancer. We showed here that tocopherol-associated protein (TAP), a vitamin E-binding protein, promoted vitamin E uptake and facilitated vitamin E antiproliferation effect in prostate cancer cells. Interestingly, without vitamin E treatment, overexpression of TAP in prostate cancer cells significantly suppressed cell growth; knockdown of endogenous TAP by TAP small interfering RNA (siRNA) in nonmalignant prostate HPr-1 cells increased cell growth. Further mechanism dissection studies suggested that the tumor suppressor function of TAP was via down-regulation of phosphoinositide 3-kinase (PI3K)/Akt signaling, but not by modulating cell cycle arrest or androgen receptor signaling. Immunoprecipitation results indicated that TAP inhibited the interaction of PI3K subunits, p110 with p85, and subsequently reduced Akt activity. Constitutively active Akt could negate the TAP-suppressive activity on prostate cancer cell growth. Moreover, stable transfection of TAP in LNCaP cells suppressed LNCaP tumor incidence and growth rate in nude mice. Furthermore, TAP mRNA and protein expression levels were significantly down-regulated in human prostate cancer tissue samples compared with benign prostate tissues as measured by reverse transcription-PCR, in situ hybridization, and immunohistochemistry. Together, our data suggest that TAP not only mediates vitamin E absorption to facilitate vitamin E antiproliferation effect in prostate cancer cells, but also functions like a tumor suppressor gene to control cancer cell viability through a non-vitamin E manner. Therefore, TAP may represent a new prognostic marker for prostate cancer progression.Item Open Access Transient Receptor Potential Vanilloid 4 Ion Channel Functions as a Pruriceptor in Epidermal Keratinocytes to Evoke Histaminergic Itch.(J Biol Chem, 2016-05-06) Chen, Yong; Fang, Quan; Wang, Zilong; Zhang, Jennifer Y; MacLeod, Amanda S; Hall, Russell P; Liedtke, Wolfgang BTRPV4 ion channels function in epidermal keratinocytes and in innervating sensory neurons; however, the contribution of the channel in either cell to neurosensory function remains to be elucidated. We recently reported TRPV4 as a critical component of the keratinocyte machinery that responds to ultraviolet B (UVB) and functions critically to convert the keratinocyte into a pain-generator cell after excess UVB exposure. One key mechanism in keratinocytes was increased expression and secretion of endothelin-1, which is also a known pruritogen. Here we address the question of whether TRPV4 in skin keratinocytes functions in itch, as a particular form of "forefront" signaling in non-neural cells. Our results support this novel concept based on attenuated scratching behavior in response to histaminergic (histamine, compound 48/80, endothelin-1), not non-histaminergic (chloroquine) pruritogens in Trpv4 keratinocyte-specific and inducible knock-out mice. We demonstrate that keratinocytes rely on TRPV4 for calcium influx in response to histaminergic pruritogens. TRPV4 activation in keratinocytes evokes phosphorylation of mitogen-activated protein kinase, ERK, for histaminergic pruritogens. This finding is relevant because we observed robust anti-pruritic effects with topical applications of selective inhibitors for TRPV4 and also for MEK, the kinase upstream of ERK, suggesting that calcium influx via TRPV4 in keratinocytes leads to ERK-phosphorylation, which in turn rapidly converts the keratinocyte into an organismal itch-generator cell. In support of this concept we found that scratching behavior, evoked by direct intradermal activation of TRPV4, was critically dependent on TRPV4 expression in keratinocytes. Thus, TRPV4 functions as a pruriceptor-TRP in skin keratinocytes in histaminergic itch, a novel basic concept with translational-medical relevance.Item Open Access Wnt signaling suppresses MAPK-driven proliferation of intestinal stem cells.(The Journal of clinical investigation, 2018-08) Kabiri, Zahra; Greicius, Gediminas; Zaribafzadeh, Hamed; Hemmerich, Amanda; Counter, Christopher M; Virshup, David MIntestinal homeostasis depends on a slowly proliferating stem cell compartment in crypt cells, followed by rapid proliferation of committed progenitor cells in the transit amplifying (TA) compartment. The balance between proliferation and differentiation in intestinal stem cells (ISCs) is regulated by Wnt/β-catenin signaling, although the mechanism remains unclear. We previously targeted PORCN, an enzyme essential for all Wnt secretion, and demonstrated that stromal production of Wnts was required for intestinal homeostasis. Here, a PORCN inhibitor was used to acutely suppress Wnt signaling. Unexpectedly, the treatment induced an initial burst of proliferation in the stem cell compartment of the small intestine, due to conversion of ISCs into TA cells with a loss of intrinsic ISC self-renewal. This process involved MAPK pathway activation, as the proliferating cells in the base of the intestinal crypt contained phosphorylated ERK1/2, and a MEK inhibitor attenuated the proliferation of ISCs and their differentiation into TA cells. These findings suggest a role for Wnt signaling in suppressing the MAPK pathway at the crypt base to maintain a pool of ISCs. The interaction between Wnt and MAPK pathways in vivo has potential therapeutic applications in cancer and regenerative medicine.Item Open Access β-arrestin 2-dependent activation of ERK1/2 is required for ADP-induced paxillin phosphorylation at Ser(83) and microglia chemotaxis.(Glia, 2012-09) Lee, Sang-Hyun; Hollingsworth, Ryan; Kwon, Hyeok-Yil; Lee, Narae; Chung, Chang YMicroglia play crucial roles in increased inflammation in the central nervous system upon brain injuries and diseases. Extracellular ADP has been reported to induce microglia chemotaxis and membrane ruffle formation through P2Y(12) receptor. In this study, we examined the role of ERK1/2 activation in ADP-induced microglia chemotaxis. ADP stimulation increases the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and paxillin phosphorylation at Tyr(31) and Ser(83) . Inhibition of ERK1/2 significantly inhibited paxillin phosphorylation at Ser(83) and the retraction of membrane ruffles, causing inefficient chemotaxis. Close examination of dynamics of focal adhesion (FA) formation with green fluorescent protein-paxillin revealed that the disassembly of FAs in U0126-treated cells was significantly impaired. Depletion of β-Arrestin 2 (β-Arr2) with short hairpin RNA markedly reduced the phosphorylation of ERK1/2 and Pax/Ser(83) , indicating that β-Arr2 is required for ERK1/2 activation upon ADP stimulation. A large fraction of phosphorylated ERK1/2 and β-Arr2 were translocated and co-localized at focal contacts in the newly forming lamellipodia. Examination of kinetics and rate constant of paxillin formation and disassembly revealed that the phosphorylation of paxillin at Tyr(31) by c-Src appears to be involved in adhesion formation upon ADP stimulation while Ser(83) required for adhesion disassembly.