1. km23: a Novel Protein in the TGF  Signaling Pathway Nicole Dague Department of Biological Sciences, York College of Pennsylvania ABSTRACT An innovative method for screening expression libraries for TGF  interacting proteins was developed by Dr. Kathleen M. Mulder at the Pennsylvania State University College of Medicine. The screening method isolated a novel TGF  receptor-interacting protein named km23. Research was conducted to determine if km23 was present with TGF  intracellularly and if km23 was phosphorylated as a means of initiating signaling. Western blots served as controls for each experiment. Experimentation confirmed that km23 was present with TGF  intracellularly and that km23 was phosphorylated by TGF . Further experimentation is necessary to determine km23’s exact role in the TGF  signaling pathway. INTRODUCTION Cancer is one of the top afflictions of Americans today. In the year 2002, 1,284,900 new cancer cases are expected to be diagnosed (American Cancer Society 2002). Transforming growth factor  (TGF  ) plays an essential role in the development of cancers and has become a popular target for research. TGF  functions as a natural potent growth inhibitor of solid tumor formation of a number of cell types. Past research has demonstrated that TGF  initiates cell signaling by phosphorylating intracellular proteins. TGF  is known to interact with two receptors on the cell surface, TGF  Receptor I (RI) and TGF  Receptor II (RII). (See Figure 1) Known molecular weights for the receptors have been published: RI ranges from 65-70kDa and RII from 85- 110kDa (Yue and Mulder 2001). An alteration in the expression of TGF  or in the proteins of its signaling pathways has been associated with human diseases (Yue and Mulder 2001). Since TGF  has been demonstrated to participate in signaling pathways that have been implicated in human pathologies, understanding it’s vital role in the development of cancer may be the key to finding a cure. Dr. Mulder’s method for screening expression libraries has isolated a novel protein involved in the TGF  - signaling pathway, named km23. Initial experimentation on km23 determined it to be a 96 amino acid protein, encoded by a 291 base pair reading frame with a predicted molecular weight of 10.6667kD. Western blotting determined actual molecular weight to be 11kD. km23 was believed to play a role in the TGF  pathway and the discovery of its role became the goal of this project. Since previous research had demonstrated that TGF  phosphorylates intracellular proteins as a mechanism for initially signaling events, a key component to our research was to determine if km23 was phosphorylated by TGF . As a component in the pathway, km23 would have the potential to serve as a therapeutic and, or diagnostic target in the battle against cancers. METHODS  Cell Culture: 293T cells were chosen for experimentation. This particular cell line is epithelial and was derived from the human kidney. The cells were designed to over-express the Tumor (T) antigen, which in turn would enhance expression of an encoded protein (km23). Cell media was DMEM + 10% FBS.  Verify km23 present with TGF ß intracellularly: 293T cells transiently transfected with tagged receptors or km23 using LipofectAMINE Plus TM. Product procedures were followed. Cells were labeled with 125 I- TGF  for 4hr at 4°C. DSS cross-linking agent added for 15 min. Lysates immunoprecipitated (IP’d) with anti-flag M2 Ab. Visualized with SDS-PAGE and autoradiography Western blotting confirmed the expression of receptors and km23 in relevant lanes Repeat experimentation confirmed results.  Determine if km23 is phosphorylated by RI/RII TGF ß receptor complex: 293T cells were transiently transfected with tagged receptors or km23 In vivo phosphorylation assays conducted (Methods from Yue and Mulder 1999). 3h labeling period with [ 32 P i ], and IP’s with anti-flag Ab. Western blotting confirmed expression in relevant lanes Results were indicative of three experiments. RESULTS  km23 was present with TGF ß intracellularly (Figure 2) Top panel: total cell lysates confirm position of RI and RII (lane 2). Banding noted in Lane 5 indicates km23 present with RI/RII complex. Lower panels: Western blots for Flag, myc, and HA confirmed expression in relevant lanes  km23 was phosphorylated by the RI/RII TGF ß receptor complex (Figure 3) Top panel: Expression of km23, RI or RII alone did not result in phosphorylation (lanes 2,3,4). km23 not phosphorylated when expressed in cell (lane 2). Phosphorylation of km23 with RI/RII receptor complex (banding in lane 5). Lower panel: Western blot analysis confirmed expression of km23-flag. CONCLUSION Experimentation supported hypothesis that km23 was present with TGF  intracellularly. (See Figure 2) Banding noted in lanes where km23 was complexed with both TGF  receptors (see Lane 5). Unlabelled TGF  served as a control and no banding was expected (see Lane 6). Separate western blots confirmed the presence of each component (km23, RI, or RII) by blotting for their specific tags. (See lower panels). Further experimentation supported that km23 was phosphorylated by TGF . (See Figure 3). Banding was noted in lane 5, when km23 was complexed with the RI/RII complex. Western blots confirmed the presence of km23. Data supported the hypothesis that km23 was present with TGF  intracellularly and that km23’s phosphorylation by TGF  suggests that km23 is part of a signaling pathway. FURTHER RESEARCH Further research has been conducted since the time of initial experimentation. Additional research has concluded that once bound to the receptor complex, km23 can mediate at least one signaling event after TGF  activation and that km23 is also associated with the motor protein dynein. The eventual goal is to determine if km23 can serve as a therapeutic or diagnostic target in the battle against cancer. ACKNOWLEDGEMENTS: Dr. Kathleen M. Mulder, Professor Department of Pharmacology Pennsylvania State College of Medicine Cory Staub, Senior Research Technician Qian Tang, Research Technician Dr. Jeffery Thompson, Faculty Advisor Figure 2 km23-Flag RI-myc RII-HA unlabelled TGF  1 2 3 4 5 6 cell lysates Flag IP RII RI Flag Ab: km23 RI myc Ab: RII HA Ab: -------- +++-+++- +++-+++- -------- +---+--- ++++++++ Western Blots for Experimental Controls: 1 2 3 4 5 6 Figure 3 IP Ab: Flag 1 2 3 4 5 6 Empty Vector km23-Flag RI-V5 RII-HA +---+--- -+---+-- --+---+- ---+---+ -+++-+++ --++--++ km23 Flag Western Blot Experimental Control: 1 2 3 4 5 6 LITERATURE CITED Derynck, R. and Feng, X. 1997. TGF-β receptor signaling. Biochimica et Biophysica Acta 1333: F105-F150. Hill, C.S. 1996. Signaling to the Nucleus by Members of the Transforming Growth Factor- β (TGF β) Superfamily. Cell Signaling 8: 533-544. Tang, Q., Staub, C.M., Gao, G., Qunyan, J., Aurigemma, R., Mulder, K.M. 2002. A novel TGFß receptor- interacting protein that is also a light chain of the motor protein dynein. Molecular Biology of the Cell 13: In press. Yue, J. and Mulder, K.M. 2001. Transforming growth factor-β signal transduction in epithelial cells. Pharmacology & Therapeutics 91: 1-34. 1.TGFß binds to RII Figure 1 Figure 1: Signal transduction through TGF ß-receptor phosphorylation and activation. Model adapted from Yue and Mulder (2001). Nucleus RI 2. RII causes RI to bind 3. RI phosphorylated, signal is initiated P Signal propagated RII TGF  RII RI TGF  Figure 2: km23 is present with the RI/RII immunocomplexes intracellularly. Figure 3: km23 is phosphorylated by the RI/RII complex.