Other chemicals were of analytical grade. Cell Culture and Preparation of Cell Extracts GT1C7 cells were kindly provided by Dr. for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer. and have been identified as important susceptibility genes for schizophrenia (8,C12). To elucidate the roles of NRG1 and ErbB4 in disease processes, it is extremely important to understand the molecular mechanisms involved in the regulation of ErbB4 in cell systems. In the previous study (13), we found that GT1C7 cells expressed ErbB4 as well as EGFR, and that transactivation of both EGFR and ErbB4 was involved in the GnRH-induced activation of ERK in the cells. In addition, we found that GnRH treatment induced the cleavage of ErbB4 (13). Pretreatment of GT1C7 cells with GnRH completely inhibited ERK activation by NRG1 treatment, indicating that GnRH treatment induced the desensitization of ErbB4 via cleavage of the protein. In the present study, we examined in detail the signal transduction mechanisms for the activation of ERK and the cleavage of ErbB4 after GnRH treatment in GT1C7 cells. The pharmacological and knockdown experiments suggested that protein kinase D (PKD) was activated by isoforms of a novel type of protein cIAP1 Ligand-Linker Conjugates 11 kinase C (novel PKC), and that PKD was involved in ERK activation but not ErbB4 cleavage. We found that Src and Fyn were constitutively activated in GT1C7 cells, whereas they activated Pyk2 only after GnRH treatment. Notably, it was interesting that PKD was necessary for the activation of Pyk2 by Src and Fyn. These results strongly suggested that PKD was involved in signal transduction between the PKC pathway and the tyrosine kinase pathway. Experimental Procedures Materials The following chemicals and reagents were obtained from the indicated sources: fetal calf serum from HyClone (Logan, UT); des-Gly10, (d-Ala6)-LH-RH ethylamide (GnRH), poly-l-lysine, mouse IgG, anti-ERK antibody (M5670), and phosphate-buffered saline from Sigma; DynaMarker Protein MultiColor from BioDynamics Lab. (Tokyo, Japan); Dulbecco’s modified Eagle’s medium from Nissui Pharmaceutical Co. (Tokyo, Japan); protease inhibitor (PI) mixture and protein phosphatase inhibitor (PPI) mixture (EDTA free) from Nacalai Tesque (Kyoto, Japan); anti-ErbB4 antibody (number 4795), anti-Src antibody (number 2108), anti-phospho-Src family (Tyr416) antibody (number 2101), anti-Fyn antibody (number 4023), anti-PKC isoform antibody sampler kit (number 9960), anti-PKD1 antibody (number 2052), anti-phospho-PKD (Ser744/748) antibody (number 2054), anti-PKD2 antibody (number 8188), anti-PKD3 antibody (number 5655), and anti-Pyk2 antibody (number 3292) from Cell cIAP1 Ligand-Linker Conjugates 11 Signaling Tecnologies (Danvers, MA); anti-Fyn antibody (ab1881) from Abcam (Cambridge, UK); anti-PKD1 antibody (A20) (sc-638) and anti-phospho-Pyk2 (Tyr402) antibody (sc-101790) from Santa Cruz, (Santa Cruz, CA); monoclonal anti-EGFR antibody (6F1) (ADI-CSA-330-E) from Assay Designs (Ann Arbor, MI); anti-PKC? antibody (GTX109028), anti-glyceraldehyde-3-phosphate cIAP1 Ligand-Linker Conjugates 11 dehydrogenase (GAPDH) antibody (GTX100118), and anti-Gq antibody (GTX104544), anti-G11 antibody (GTX118876) from GeneTex Inc. (San Antonio, TX); NF449 from Calbiochem (Darmstadt, Germany); pertussis toxin from Seikagaku Biobusiness Corp. (Tokyo, Japan); bisindolylmaleimide I from Enzo Life Science cIAP1 Ligand-Linker Conjugates 11 (Farmingdale, NY); dasatinib from BioBision (Milpitas, CA); G? 6976 and CRT0066101 from Tocris Bio. (Minneapolis, MN); anti-active ERK antibody (V8031) and phorbol 12-myristate 13-acetate (PMA) from Promega Corp. (Madison, WI); and SDS-PAGE molecular weight standards from Bio-Rad. YM-254890 was generously provided by Taiho Pharmaceutical Co., Ltd. (Tokyo, Japan) (14). Other chemicals were of analytical grade. Cell Culture and Preparation of Cell Extracts GT1C7 cells were kindly provided by Dr. R. Weiner (University of California) and Dr. M. Kawahara (Musashino University, Japan) (15, 16). The cells were grown on 0.02% (w/v) poly-l-lysine-coated Petri dishes (Nunc, Roskilde, Denmark) as described previously (17). We chose the concentrations of signal transduction inhibitors (NF449, pertussis toxin, YM-254890, bisindolylmaleimide I, G? 6976, CRT0066101, dasatinib, PP2, and Src Inhibitor 1) as directed by cIAP1 Ligand-Linker Conjugates 11 the manufacturer’s. Cells were lysed in 1 SDS-PAGE sample buffer containing 2% (w/v) SDS, 62.5 FASN mm Tris-HCl, pH 6.8, 5% (v/v) 2-mercaptoethanol, 5% (v/v) glycerol, and 0.01% (w/v) bromphenol blue (18). The cell lysate.