ARTHRITIS & RHEUMATISM Vol. 63, No. 7, July 2011, pp 1833–1842 DOI 10.1002/art.30375 © 2011, American College of Rheumatology Tyr 323 -Dependent p38 Activation Is Associated With Rheumatoid Arthritis and Correlates With Disease Activity Mercedes Lo ´pez-Santalla, 1 Marı ´a Salvador-Berna ´ldez, 1 Isidoro Gonza ´lez-Alvaro, 2 Santos Castan ˜eda, 2 Ana M. Ortiz, 2 Marı ´a Isabel Garcı ´a-Garcı ´a, 1 Leonor Kremer, 1 Fernando Roncal, 1 Juan Mulero, 3 Carlos Martı ´nez-A, 1 and Jesu ´s M. Salvador 1 Objective. The p38 MAPK is important in the pathogenic immune response in rheumatoid arthritis (RA). The p38 molecule can be activated through phos- phorylation on Thr 180 –Tyr 182 by upstream MAPK ki- nases and via an alternative pathway through phosphor- ylation on Tyr 323 . We undertook this study to quantify the phosphorylation of Tyr 323 p38 and of Thr 180 –Tyr 182 p38 on T cells from healthy controls and patients with RA or ankylosing spondylitis (AS) to identify vari- ables associated with p38 phosphorylation and disease activity. Methods. We measured p38 phosphorylation on Tyr 323 and Thr 180 –Tyr 182 by flow cytometry and Western blotting on T cells from 30 control subjects, 33 AS patients, 30 patients with RA in remission, and 79 patients with active RA. We collected the clinical char- acteristics and analyzed correlations between clinical variables, the Disease Activity Score in 28 joints (DAS28), and p38 phosphorylation levels. Multivariate regression analysis was performed to identify variables associated with p38 phosphorylation on Tyr 323 and Thr 180 –Tyr 182 . Results. Phosphorylation of p38 on Tyr 323 was higher in T cells from patients with active RA (P  0.008 versus healthy controls) than in patients with RA in remission or in patients with AS. Tyr 323 p38 phosphor- ylation was associated with disease activity determined by the DAS28 (P  0.017). Enhanced p38 phosphoryla- tion was linked to Lck-mediated activation of the Tyr 323 - dependent pathway in the absence of upstream MAPKK activation. Conclusion. Our results indicate that phosphor- ylation status on Tyr 323 p38 correlates with RA disease activity and suggest that the Tyr 323 -dependent pathway is an attractive target for down-regulation of p38 activ- ity in RA patients. MAPKs comprise 4 subfamilies: p38, ERK, JNK, and ERK-5 (1–4). The p38 MAPK participates in many cell functions such as cell cycle regulation (5), prolifer- ation (6), differentiation (7), apoptosis (8), and devel- opment (9), as well as the Th1 immune response asso- ciated with rheumatoid arthritis (RA) progression (10,11). The activity of p38 is induced by stress- associated stimuli (12,13) and by proinflammatory cyto- kines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor  (TNF) (14,15). Although p38 activa- tion is implicated in cytokine release and inflammatory processes that cause disease progression in RA (16–21), the mechanisms involved in this activation have not been established. In mammalian cells, the best-characterized mech- anism for p38 activation is by way of a phosphorylation Supported by the European Union (Sixth Framework Marie Curie Programme grant MIRG-CT-2006-036592, and Seventh Frame- work Programme integrated project Masterswitch grant Health-F2- 2008-223404 and European Research Council Starting Grant FP7- 208250-1), the Spanish Ministry of Health (grants PI052731 and PI081718), the Spanish Ministry of Science and Innovation (grant 200820I109), the Madrid Regional Government (grant 200520M122), and the Genoma Espan ˜a Foundation. Dr. Lo ´pez-Santalla is recipient of a fellowship from the Spanish Ministry of Health. Ms Salvador- Berna ´ldez is recipient of fellowships from the Spanish Ministry of Health and from the Spanish Ministry of Science and Innovation through the JAE-CSIC Programme. 1 Mercedes Lo ´pez-Santalla, PhD, Marı ´a Salvador-Berna ´ldez, Marı ´a Isabel Garcı ´a-Garcı ´a, Leonor Kremer, PhD, Fernando Roncal, PhD, Carlos Martı ´nez-A, PhD, Jesu ´s M. Salvador, PhD: Centro Nacional de Biotecnologı ´a, CSIC, Madrid, Spain; 2 Isidoro Gonza ´lez- Alvaro, MD, PhD, Santos Castan ˜eda, MD, PhD, Ana M. Ortiz, MD, PhD: Hospital Universitario de la Princesa IIS-Princesa, Madrid, Spain; 3 Juan Mulero, MD, PhD: Hospital Puerta de Hierro, Madrid, Spain. Address correspondence to Jesu ´s M. Salvador, PhD, Depart- ment of Immunology and Oncology, Centro Nacional de Biotecnologı ´a, CSIC, Darwin 3, Campus de Cantoblanco, E-28049 Madrid, Spain. E-mail: jmsalvador@cnb.csic.es. Submitted for publication April 27, 2010; accepted in revised form March 22, 2011. 1833