Abstract
Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr(160) phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr(152-156), Tyr(567), and a proline-rich region, Pro(415-417). Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.
Highlights
NPM-anaplastic lymphoma kinase (ALK)-induced signaling pathways control cellular proliferation, inhibit apoptosis, and modify cell-cell adhesion and migration [3,4,5,6] and cytoskeleton reorganization and transformation [6, 7]
We transfected human HEK-293T with ALKFL, with NPM-ALK (which is both cytoplasmic and nuclear [27]), with its kinase-dead mutant (K210R), with ATIC-ALK, and with a chimeric fusion protein between CD8 and ALK (CD8-ALK) that localizes to the cytoplasmic membrane
We have characterized the interaction of the adaptor protein Grb2 with NPM-ALK, the fusion protein involved in the pathogenesis of the anaplastic large cell lymphoma
Summary
NPM-ALK-induced signaling pathways control cellular proliferation, inhibit apoptosis, and modify cell-cell adhesion and migration [3,4,5,6] and cytoskeleton reorganization and transformation [6, 7]. Adaptor proteins Shc (SH2 domain-containing transforming protein), IRS-1 (insulin receptor substrate 1), and Grb bind NPM-ALK directly or in complexes, but their specific role in NPM-ALK-mediated lymphomagenesis is still unclear [2, 8]. The SH2 domain of Grb binds the tyrosine-phosphorylated peptide sequences of receptor tyrosine kinases such as the EGF receptor [14], PDGF receptor [12], and T-cell receptor [15] and of oncogenic fusions such as BCR-ABL [16] and TPR-MET [17]. We found that Tyr160 of Grb is phosphorylated by other oncogenic fusion tyrosine kinases such as TPR-MET, BCR-ABL, and TEL-JAK2, as well as by wild-type receptor tyrosine kinases such as ALK and MET. Grb in NPM-ALK Signaling that Grb is essential for the activation of SHP2 in ALCL and is required for sustained ALCL cell growth
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