[CANCER RESEARCH 59, 1192–1195, March 15, 1999] Advances in Brief Functional Interaction between Retinoblastoma Protein and Stress-activated Protein Kinase in Multiple Myeloma Cells 1 Dharminder Chauhan, Teru Hideshima, Steve Treon, Gerrard Teoh, Noopur Raje, Shima Yoshihimito, Yu-Tzu Tai, Weiwei Li, Jianguo Fan, James DeCaprio, and Kenneth C. Anderson 2 Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 [D. C., T. H., S. T., G. T., N. R., S. Y., Y-T. T., J. D., K. C. A.], and Program for Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [W. L., J. F.] Abstract Previous studies have demonstrated that -irradiation (IR)-induced apop- tosis in multiple myeloma (MM) is associated with activation of stress- activated protein kinase (SAPK). In the present study, we examined the molecules downstream of SAPK/C-Jun N-terminal kinase (JNK), focusing on the role of retinoblastoma protein (Rb) during IR-induced MM cell apoptosis. The results demonstrate that IR activates SAPK/JNK, which associates with Rb both in vivo and in vitro. Far Western blot analysis confirms that SAPK/ JNK binds directly to Rb. IR-activated SAPK/JNK phosphorylates Rb, and deletion of the phosphorylation site in the COOH terminus domain of Rb abrogates phosphorylation of Rb by SAPK/JNK. Taken together, our results suggest that Rb is a target protein of SAPK/JNK and that the association of SAPK/JNK and Rb mediates IR-induced apoptosis in MM cells. Introduction Treatment of eukaryotic cells with IR 3 induces growth arrest, activation of DNA repair, and apoptosis. The finding that treatment with IR induces transcription of early response genes, such as c-jun and Egr-1, suggests the involvement of nuclear events (1, 2). To date, multiple protein kinases have been identified that transduce IR- induced signals to the nucleus. SAPK, also known as JNK, is related to the mitogen-activated protein kinases (3–7) and has been linked to apoptosis (8). Two serine residues (Ser 63 and Ser 73 ) in the NH 2 - terminal transactivation domain of c-Jun have been identified as substrates for SAPK/JNK (6, 7). Previous studies have shown that stress stimuli, e.g., IR, tumor necrosis factor, sphingomyelinase, and UV light, activate SAPK/JNK (6 –10). Our recent studies demonstrate that IR- and Fas-induced activation of SAPK/JNK in MM cells is associated with apoptosis (11, 12). To date, however, the mechanism whereby SAPK/JNK transduces apoptotic signals during MM cell apoptosis remains largely unknown. The Rb protein is a nuclear phosphoprotein that regulates cell growth in the G 1 phase of the cell cycle (13, 14). Phosphorylation of Rb plays an important role in its growth-inhibitory function by mod- ulating its interaction with other proteins, such as E2F (13, 14). Recent studies have also suggested a role for Rb in apoptosis (14, 15), in particular, during IR-induced apoptosis (16). Rb-mediated apoptosis has been linked to activation of serine proteases (14, 15). In addition, Rb protein may be a target of proteolysis by caspases, further sug- gesting its potential role during apoptosis (17). In the present study, we characterized the signaling mechanisms during IR-induced apoptosis in a MM-derived cell line. The results demonstrate that IR-activated SAPK/JNK binds directly to Rb and phosphorylates Rb, suggesting that SAPK/JNK-Rb mediates IR- induced apoptosis in MM cells. Materials and Methods Cell Culture and Metabolic Labeling. The human MM cell line OCI- MY5 and Rb-deficient (Rb-/-) SAOS-2 and Rb-reconstituted (Rb+/+) SAOS-2 cell lines were cultured, and -IR was performed as described previously (11, 18). For labeling, cells (1  10 7 ) were resuspended in 1 ml of phosphate-free DMEM and incubated for 1 h before the addition of 5 mCi of [ 32 P]orthophosphate (carrier-free; ICN). The cells were then incubated for 5 h, treated with IR for 1 h, and harvested. Lysates were then subjected to immu- noprecipitation with anti-Rb Ab. Reagents and Abs. Anti-SAPK/JNK and anti-tubulin Abs were purchased from Santa Cruz Biotechnology (San Diego, CA); anti-Rb mAb (G3-245) was obtained from PharMingen (San Diego, CA). The GST-Rb (full length, 379 – 928) and various GST-Rb deletion constructs were kind gifts of Drs. William G. Kaelin, Jr. and Peter D. Adams (Dana-Farber Cancer Institute, Boston, MA). Immunoblotting/Immunoprecipitation. Cell lysates were prepared as de- scribed previously (11, 12, 19). Equal amounts of proteins (250 –300 g) were subjected to immunoprecipitation with the indicated Abs, and immune com- plexes were precipitated with protein A-Sepharose. The resulting protein precipitates were washed three times with lysis buffer and resolved by SDS- PAGE. Proteins were then transferred to nitrocellulose filters, blocked by incubation in 5% dry milk in PBST (0.05% Tween-20 in PBS), and probed with the indicated antibodies. Blots were then developed by enhanced chemi- luminesence (Amersham, Arlington Heights, IL). In Vitro Immune Complex Assays. Cell lysates were incubated with either PIRS or SAPK/JNK Abs for 2 h at 4°C before the addition of protein A-Sepharose. The immune complexes were washed three times with lysis buffer and once with kinase buffer and resuspended in kinase buffer containing [- 32 P]ATP (3000 Ci/mmol; New England Nuclear, Boston, MA) and GST- Jun (2–100) or GST-Rb deletion constructs as substrates. The reaction was incubated for 15 min at 30°C and terminated by the addition of SDS sample buffer. Proteins were analyzed by SDS-PAGE, Coomassie Blue staining, and autoradiography. Far Western Analysis. The lysates from IR-treated cells were subjected to immunoprecipitation with anti-SAPK Ab and PIRS. The proteins were re- solved by 10% SDS-PAGE and transferred to nitrocellulose membrane. The filters were first incubated with purified GST-Rb (full length, 379 –928) fusion protein (19) or with purified GST fusion protein alone (negative control) at room temperature for 2 h and then analyzed by immunoblotting with anti-GST mAb. Results and Discussion IR Induces SAPK/JNK Activation and Formation of SAPK/ JNK-Rb Complexes. Many studies have demonstrated that IR in- duces apoptosis in various cell types. We and others have demon- Received 1/12/99; accepted 2/1/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by USPHS Grant CA50947 to (K. C. A.) and a Kathy Giusti Multiple Myeloma Research Foundation Fellowship to (D. C.). 2 To whom requests for reprints should be addressed, at Department of Adult Oncol- ogy, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02215. Phone: (617) 632-2144; Fax: (617) 632-2569; E-mail: kenneth_anderson@dfci.harvard.edu. 3 The abbreviations used are: IR, irradiation; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; Rb, retinoblastoma; MM, multiple myeloma; Ab, antibody; GST, glutathione S-transferase; mAb, monoclonal Ab; PIRS, preimmune rabbit serum. 1192 Research. on October 10, 2021. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from