Role of the phosphatase PP4 in the activation of JNK-1 in prostate carcinoma cell lines PC-3 and LNCaP resulting in increased AP-1 and EGR-1 activity

INOSTROZA ET AL. Biol Res 38, 2005, 163-178 Biol Res 38: 163-178, 2005 BR163 Role of the phosphatase PP4 in the activation of JNK-1 in prostate carcinoma cell lines PC-3 and LNCaP resulting in increased AP-1 and EGR-1 activity JUAN INOSTROZA1, LEONARDO SÁENZ1, GLORIA CALAF2, GERTRUDIS CABELLO2 and EDUARDO PARRA1 1 Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile. 2 Departamento de Biología y Salud. Facultad de Ciencias, Universidad de Tarapacá, Arica, Chile. ABSTRACT The specific signaling connections between the mitogen-activated protein kinases (MAPK) such as c-Jun Nterminal kinase (JNK-1) and phosphatases PP4 and M3/6, affecting the family of early nuclear factors, is complex and remains poorly understood. JNK-1 regulates cellular differentiation, apoptosis and stress responsiveness by up-regulating early nuclear factors such as c-Jun, a member of the activating protein (AP1) family, and the Early Growth Factor (EGR-1). C-Jun, when phosphorylated by c-Jun N-terminal kinase (JNK-1) associates with c-Fos to form the AP-1 transcription factor that activates gene expression. We have investigated the regulation of the JNK-1 kinase by co-transfecting phosphatases PP4 and M3/6 in prostate cancer cell lines PC-3 and LNCaP, which have been previously stimulated with human EGF or cisplatin. Cotransfections of plasmids expressing the JNK-1 and the serine/threonine phosphatases PP4 resulted in a significant increase in JNK-1 activity in both PC3 and LNCaP cells. In contrast, co-transfection of JNK-1 with the dual specific phosphatase serine/threonine M3/6 showed only a marginal effect in JNK-1 activity. The phosphatase M3/6 also failed in blocking the induction of JNK-1 activity observed in presence of PP4. The higher activity of JNK-1 was associated with increased activities of the factors c-Jun/AP-1 and EGR-1. This suggests that JNK-1 activity in PC-3 and LNCaP cells requires not only active PP4 for stable maintenance but also suggests that the relative degree of phosphorylation of multiple cellular components is the determinant of JNK-1 stability. Key terms: prostate cancer, phosphatase PP4, kinase JNK-1 INTRODUCTION The MAPK family consists of a group of three different kinase pathways: the extracellular signal-regulated kinases (ERK1/2), a.k.a. p44/42 (Robinson et al., 1996); the Jun-N-terminal kinases (JNKs), a.k.a. SAPK (stress-activated protein kinases) (Blenis, 1993; Cobb et al., 1995); and the p38 kinase, which is usually activated by environmental stresses and correlated with programmed cell death (Ip & Davis, 1998; Derijard et al., 1994; Foltz et al., 1998; Hibi et al., 1993). Recently, an important role of the JNK-1/2 member of the MAP kinases has been proposed in prostate cancer. JNK-1 is activated by dual phosphorylation of Thr and Tyr residues within a Thr-Pro-Tyr motif located in kinase sub domain VIII (Sánchez et al., 1994; Tournier et al., 1997). Once activated, JNK-1 can up-regulate gene expression by phosphorylating the activating motif of transcription factors such as ATF2 (Gupta et al., 1995; Bocco et al., 1996), cJun and JunD (Hibi et al., 1993; GroverBardwick, et al., 1994), and the Ets domain of transcription factors Elk-1 and Sap-1 (Minden et al., 1994; Smeal et al., 1992; Janknecht et al., 1993; Janknecht and Hunter 1997). The activation of the transcription Corresponding author: Eduardo Parra, Ph.D. Signal Transduction and Gene Therapy Lab, Disciplinary Program of Pharmacology, ICBM. Faculty of Medicine, Universidad de Chile, Av. Independencia 1027, Santiago, Chile, Tel.: (56-2) 978-6654, Fax: (56-2) 737-2783, E-mail: Received: October 19, 2004. In revised form: June 21, 2005. Accepted: June 23, 2005. 164 INOSTROZA ET AL. Biol Res 38, 2005, 163-178 factor AP-1 mediated by JNK-1, by means of phosphorylation of c-Jun, has been implicated in oncogenic transformation (Karin, 1995). Transcription factor phosphorylation by protein kinases has received a great deal of attention in the study of prostate cancer, in contrast, very little is known about the implications of protein phosphatases (PP) and de-phosphorylation in this type of cancer. The phosphorylation of MAP kinases (MAPK) in vivo is a reversible process, indicating that protein phosphatases provide an additional level of regulation of MAPKs functions (Mustelin et al., 2005). A group of dual-specificity kinase phosphatases, (MAPs), has been identified that inactivates MAPKs through dephosphorylation of both threonine and tyrosine residues (Guan et al., 1991). These phosphatases include ERK-specific MKP-1, -2 and -3 (Fanger, 1997; Haneda et al., 1999; Keyse, 1998) and JNK-1/2 and p38-specific M3/6 (Muda et al., 1996). There is growing evidence suggesting that protein phosphatases are implicated in prostate cancer. The phosphatase PTEN, also a dual specificity protein phosphatase, is inactivated in a significant proportion of prostate carcinoma, which suggests that its inactivation is associated with the metastatic stage of prostate cancer (Yen et al., 1997; Vliestra et al., 1998). The mitogen-activated protein kinase 1 phosphatase (MKP-1) is over-expressed in the pre-invasive stage of prostate cancer, showing decrease levels in the advanced stage of the disease, suggesting that over-expression of MKP-1 could be associated with the early phase of prostate cancer (Muda et al., 1996) The phosphatase PP4 (also called PPX), a novel serine/threonine phosphatase, seems to play a key role in the regulation of JNK-1 expression. PP4 is structurally related to the PP2A family of enzymes (with 65% amino acid sequence identity) and contains putative okadaic acid and microcystin-LR binding domains (Cohen, 1997; Shiozaki & Russell, 1995). PP4 has been highly conserved during evolution (Cohen, 1990; 1997). The deduced amino acid sequence of human PP4, assigned to chromosome 16p11-p12, differs from the sequence of rabbit PP4 only in two of 307 amino acids (Cohen, 1990; 1997). This high degree of conservation suggests that PP4 is involved in critical cellular events. Since PP4 is found predominantly in centrosomes, microtubule nucleation could be one of such event (Shiozaki and Russell, 1995; Zhou et al., 2002). However, the molecular mechanisms by which PP4 functions in cellular signaling remain to be determined. Another factor that is overexpressed in prostate cancer is the Early Growth Response-1 (EGR-1), which is a member of the immediate-early gene family that includes Fos, Jun, and other early growth response genes (You et al., 1997; Mora et al., 2004; Baron et al., 2003). EGR1 gene encodes a DNA-binding protein that contains zinc finger motifs, expressed in many cell types in response to a wide variety of mitogenic and non-mitogenic stimuli, including peptide growth factors, shear stress, urea and hypotonicity (Pipaon et al., 2004; Keeton et al., 2003). Once activated, EGR-1 binds to 5'-GCGGGGGCG-3' consensus sequences within the promoter region of (...truncated)