Tnk1/Kos1: a novel tumor suppressor

Abstract

Tnk1/Kos1 is a non-receptor protein tyrosine kinase implicated in negative regulation of cell growth by a mechanism involving inhibition of Ras activation and requiring Tnk1/Kos1's intrinsic catalytic activity. Tnk1/Kos1 null mice were created by homologous recombination by deleting the catalytic domain. Upon aging, both Tnk1+/- and Tnk1-/- mice develop spontaneous tumors, including lymphomas and carcinomas at high rates (i.e. 27%, and 43%, respectively), indicating that Tnk1/Kos1 is a tumor suppressor. Tissues from Tnk1/Kos1-null mice exhibit proportionally higher levels of basal and growth factor-stimulated Ras activation. Mechanistically, Tnk1/Kos1 requires either or both Y277 and Y287 sites to be intact for enzymatic activity and phosphorylation of its substrate, growth factor receptor binding protein 2 (Grb2). Data indicate that following tyrosine phosphorylation of Grb2 by Tnk1/Kos1, the Grb2-Sos1 guanine exchange factor (GEF) complex that mediates growth factor stimulated Ras activation becomes disrupted, resulting in the reversal of Ras activation. Conversely, the loss of Tnk1/Kos1 activity results in constitutive activation of Ras due to prolonged stabilization/activation of the Grb2-Sos1 GEF activity. Tnk1/Kos1 is the first tyrosine kinase discovered to have tumor suppressor activity, and the mechanism of spontaneous tumor formation involves constitutive, indirect activation of Ras. Thus, Ras may display "oncogenic activity" without undergoing "oncogenic" mutation. We now find that a cohort of patients with diffuse large B-cell lymphoma (DLBCL) display downregulation of Tnk1/Kos1 that may account for tumorigenesis in humans.

Fig. 1

Spectrum of spontaneous tumors that develop in Tnk1/Kos1 knockout mice. Sixty of 140 (43%) Tnk1^−/− homozygous mice and 54 of 200 (27%) Tnk1+/− heterozygous mice developed tumors with aging. The majority of the tumors are classified as diffuse large B-cell lymphomas.

Fig. 2

High Endogenous Ras activity in Tnk1/Kos1 null mice prior to tumor development. Endogenous Ras activity in the spleen of six week old mice was determined (25,31). A dose dependent elevation of Ras activity (hyper active Ras) in spleen associated with allelic loss of Tnk1 gene in heterozygous and homozygous null mice both in the absence and presence of 100 ng/ml of Granulocyte Macrophage-Colony Stimulating Factor (GMCSF).

Fig. 3

Tnk1/Kos1 directly associates with Sos1 and Grb2 but not with Ras. GFP, GFP-Kos1 and GFP-Kos1(CN) were immuno-precipiated from BaF3 cells cell lysates (1mg) using a anti-GFP antibody (24). Western analysis of 100 μg of cell lysates was compared with the anti GFP antibody pull-downs.

Fig. 4

Grb2 is the substrate of Tnk1/Kos1. Cos7 cells were transfected with GFP, GFP-Kos1 and kinase-dead GFP-Kos1(CN). GFP and GFP fusion proteins were immuno-precipitated using an anti GFP antibody and an in vitro PTK assay was performed using 0.25 μg of recombinant Grb2 and [γ-³²P]ATP. Western analysis was performed using anti GFP, Py20, and anti Grb2 antibodies. Grb2 is tyrosine phosphorylated by GFP-Kos1, which undergo auto- tyrosine phosphorylation. GFP-only or GFP-Kos1(CN) fail to tyrosine phosphorylate Grb2 (31).

Fig. 5

Tnk1/Kos1 uncouples Grb2-Sos1 GEF complex. Receptor mediated Ras activation is regulated by guanine nucleotide exchange factors, which catalyze the exchange of GDP for GTP. The model depicts the mechanism by which activated Tnk1/Kos1 inhibits the Ras GEF activity by uncoupling the activated Grb2-Sos1 GEF complex to indirectly suppress Ras and abort downstream Ras signaling.

Fig. 6

PTK activity of Tnk1/Kos1 tyrosine (Y) to phenyl alanine (F) mutants. Panel A: The Tnk1/Kos1 point mutants (Y→F) were created by site-directed mutagenesis. Cos7 cells were transfected with GFP, GFP-Kos1 or GFP-Kos1 mutants and were immuno precipitated using an anti GFP antibody. Protein bands were resolved on a 10% SDS-PAGE and transferred on to nitro-cellulose paper. Auto tyrosine kinase activity of GFP, GFP-Kos1 or GFP-Kos1-mutants was determined using Py20. Anti-GFP antibody was used to determine the expression of GFP and the GFP fusion proteins (24). Panel B: The immunoprecipitates containing GFP-Kos1 or Kos1 (Y→F) mutants were subjected to invitro PTK assay using 0.25 μg Grb2 and cold ATP (2). The reaction mix was subjected to SDS-PAGE as above for Western analysis using the phospho-tryosine antibody Py20, anti-GFP and anti-Grb2 antibodies.