BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 232, 273–277 (1997) ARTICLE NO. RC976289 Requirement of Phosphatidylinositol-3 Kinase for Activation of JNK/SAPKs by PDGF Marco Lopez-Ilasaca,* Weiqun Li,† Aykut Uren,† Jin-chen Yu,‡ Andrius Kazlauskas,§ J. Silvio Gutkind, Ø and Mohammad A. Heidaran ,1 *Max-Planck Research Group, Faculty of Medicine, Friedrich Schiller Universita ¨ t, Drackendorfer Str. 1, D-07747 Jena, Germany; †National Institute of Cancer Research, National Institutes of Health, 9000 Rockville Pike, Building 37, Room 1E24, Bethesda, Maryland 20892-4330; ‡Cor Therapeutics, 256 East Grand Ave., South San Francisco, California 94080; §Division of Signal Transduction, Harvard Medical School, Boston, Massachusetts; Ø National Institute of Dental Research, Bldg. 30, Room 211, 9000 Rockville Pike, Bethesda, Maryland 20892; and Orquest Biotechnology, 365 Ravendale Drive, Mountain View, California 94043 Received January 27, 1997 pholipase A2, p90 Rsk and nuclear proteins such as the The molecular mechanism by which cell surface re- ternary complex factor p62 TCF and ElK-1 (2). In con- ceptors stimulate the serine/threonine kinase activity trast, JNKs phosphorylate the amino terminal trans- of c-Jun N-terminal kinases (JNKs) was investigated activating domain of c-jun and ATF2 (3), thereby in- using a transient cotransfection experiments in COS- creasing their transcriptional activity. 7 cells. Our data demonstrate that JNK activity is po- Although, recent findings have helped to unveil the tently induced by platelet derived growth factor pathway linking cell surface receptors to ERKs, the (PDGF) upon expression of bPDGFR wild type mechanism of activation of JNKs by receptor tyrosine (bRWT). However, PDGF failed to mediate JNK activa- kinases (RTKs) is less well defined (1,4). Platelet-de- tion in cells expressing bPDGFR mutant lacking the rived growth factor (PDGF) is a potent mitogen for binding site for phosphatidylinositol-3 (PI-3) kinase connective tissue cells (5). PDGF mediates its diverse but not for phospholipase Cg (PLCg) or Syp. Consis- biological functions by binding to two related high af- tent with this result, a PI-3 kinase inhibitor, wortman- finity receptors designated as aPDGFR and bPDGFR nin inhibited activation of JNK by PDGF. Further- (6,7). Activation of the PDGFR tyrosine kinase domain more, overexpression of P110 the catalytic domain of leads to physical association and tyrosine phosphory- PI-3 kinase was sufficient for activation of JNKs which could be efficiently inhibited by dominant negative lation of many substrates, including Src family mem- forms of Ras, Rac but not of RhoA or Cdc42. Taken bers Src, yes and fyn, the 85 kDa subunit of PI-3 ki- together all of these findings suggest that activation nase (p85), Nck, RasGTPase-activating protein (Ras- of JNK by PDGF involves receptor association with PI- GAP), phospholipase C-g (PLCg) and Syp (8-13). 3 kinase activity, which in turn acts on a ras- and rac- The specific tyrosine residues interacting with each dependent pathway.  1997 Academic Press of these substrates have been identified for the bPDGFR. Src binds to tyrosines 579 and 581 within the juxtamembrane domain (14), and p85 binds to ty- rosines 740 and 751 within the kinase insert domain of Stimulation of a variety of cell surface receptors bPDGFR (15 – 17). Tyrosine 751 is also the association causes a rapid increase in the enzymatic activity of a site for Nck (18). RasGAP binds to tyrosine 772 within family of serine/threonine kinases known as mitogen the kinase insert domain of bPDGFR (16,17). Syp and activated protein kinases (MAPKs). MAPKs have been PLCg bind to tyrosine 1009 and 1021, respectively, classified into three subfamilies: extracellular signal within the carboxy-terminal domain of bPDGFR regulated kinases (ERKs), including ERK1 and ERK2 (19,20). In addition, Shc and protein kinase C-d (PKC- also known as p44 mapk and p42 mapk , respectively; stress d) have recently been shown to be phosphorylated on activated protein kinases (SAPKs), also termed as c- tyrosine following PDGF-BB stimulation (21,22). jun N-terminus kinases (JNKs); and p38 kinase (1). Whereas, RTKs activate JNKs poorly in fibroblasts ERKs phosphorylate and regulate the activity of phos- (23), this class of receptors have been shown to induce activation of JNKs in other cells of epithelial origin (24 & unpublished observation). Thus, we sought to 1 Corresponding author. 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 273