SHP2 mediates the protective effect of interleukin-6 against dexamethasone-induced apoptosis in multiple myeloma cells.

Our previous studies have shown that activation of a related adhesion focal tyrosine kinase (RAFTK) (also known as Pyk2) is required for dexamethasone (Dex)-induced apoptosis in multiple myeloma (MM) cells and that human interleukin-6 (IL-6), a known growth and survival factor for MM cells, blocks both RAFTK activation and apoptosis induced by Dex. However, the mechanism whereby IL-6 inhibits Dex-induced apoptosis is undefined. In this study, we demonstrate that protein-tyrosine phosphatase SHP2 mediates this protective effect. We show that IL-6 triggers selective activation of SHP2 and its association with RAFTK in Dex-treated MM cells. SHP2 interacts with RAFTK through a region other than its Src homology 2 domains. We demonstrate that RAFTK is a direct substrate of SHP2 both in vitro and in vivo, and that Tyr(906) in the C-terminal domain of RAFTK mediates its interaction with SHP2. Moreover, overexpression of dominant negative SHP2 blocked the protective effect of IL-6 against Dex-induced apoptosis. These findings demonstrate that SHP2 mediates the anti-apoptotic effect of IL-6 and suggest SHP2 as a novel therapeutic target in MM.

Our recent study demonstrated that RAFTK mediates Dex-induced apoptosis in human multiple myeloma (MM) cells (10). Importantly, Interleukin-6 (IL-6) inhibits both Dex-induced RAFTK activation and apoptosis in these cells. However, the molecular mechanisms by which IL-6 inhibits Dex-induced apoptosis are undefined.
Interleukin-6 binds to its receptor (IL-6R/gp80) which interacts with the signal transducer gp130. The cytoplsmic domain of gp130 contains phosphotyrosine motifs for recruitment of a protein tyrosine phosphatase SHP2, also referred to as SHPTP2 (11,12).
SHP2 is widely expressed and is required as a positive (i.e., signal enhancing) component of growth factor and cytokine signal transduction pathways (13)(14)(15). For example, our prior studies have demonstrated that IL-6 triggers proliferation of MM cells via by guest on  http://www.jbc.org/ Downloaded from the mitogen activaed protein kinase (MAPK) cascade, which includes SHP2 activation (16). Moreover, fibroblasts from SHP2 mutant mice exhibit impaired MAPK activation in response to fibroblast growth factor, epidermal growth factor, and insulin growth factor (17,18). Although SHP2 may directly bind and dephosphorylate cytokine receptors or other cytoplasmic protein tyrosine kinases (14,(19)(20), its role in regulating apoptotic signaling is at present undefined.
The present study examined the role of SHP2 in mediating the inhibitory effects of IL-6 on Dex-induced apoptosis. We demonstrate that IL-6 activates SHP2 in MM cells, and that treatment of MM cells with IL- 6
Immunoprecipitation and immunoblot analysis Cells were washed with PBS and lysed in 1 ml of lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM sodium vanadate, 1 mM PMSF, 1 mM DTT and 10 µg/ml of leupeptin and aprotinin) as previously described (10). Lysates were subjected to immunoprecipitation with anti- DNA-end labelling was performed as previously described (24). The mechanism whereby IL-6 inhibits Dex-induced RAFTK activation and apoptosis is unknown. We and others have shown that IL-6 induces growth in MM cells via the MAPK pathway, which includes activation of protein tyrosine phosphatase SHP2 (16,28).
To determine whether SHP2 may also mediate the inhibitory effect  Incubation of WT-RAFTK was associated with binding to wild-type SHP2 (Fig. 4A, upper panel). Reprobing the filters with anti-Flag demonstrated equal RAFTK protein levels (Fig. 4A, lower panel).
We next determined whether association of SHP2 with RAFTK is SH2dependent. A similar experiment, as described above, was Following treatment, cells were analyzed for apoptosis by DNA fragmentation and propidium iodide staining. Overexpression of SHP2, but not SHP1, blocks Dex-induced apoptosis (Fig. 6B).
To We and others have shown that IL-6 is a growth and anti-apoptotic factor for MM cells (10,16,26,27). IL-6 uses JAK-STAT and RAS- In the present study, we show that IL-6 activates SHP2, which then associates, with RAFTK in Dex-treated MM cells. SHP2 interacts with RAFTK through a region other than its SH2 domains.
We demonstrate that RAFTK is a substrate of SHP2, and that Y906 in the C-terminal domain of RAFTK mediates its interaction with SHP2.
Furthermore, overexpression of dominant negative (DN)-SHP2 blocked the protective effect of IL-6 against Dex-induced apoptosis.
Collectively, the present model suggests that the IL-6-activated SHP2 dephosphorylate RAFTK, thereby blocking Dex-induced apoptosis (Fig 7). These studies provide the framework for novel treatment approaches targeting SHP2 in MM.