Transforming growth factor-α increases tyrosine phosphorylation of microtubule-associated protein kinase in a small intestinal crypt cell line (IEC-6)

The small intestinal crypt cell line (IEC-6) is an undifferentiated, untransformed, mitotically active cell used in this study to determine the effect of transforming growth factor-alpha (TGF-alpha) on tyrosine phosphorylation levels of cellular proteins. Thymidine incorporation increased maximally after addition of 2 ng/ml TGF-alpha for 24 h. At the same dose, TGF-alpha induced the tyrosine phosphorylation of proteins with approximate molecular masses of 42, 44, 52, 80, 150 and 175 kDa as shown by Western blots treated with anti-phosphotyrosine antibody. The most intense phosphorylation was seen in the 42 kDa (p42) and 44 kDa (p44) proteins, which were identified as two isoforms of microtubule-associated protein kinase (MAPK). This phosphorylation was seen as early as 5 min post stimulation and was dose dependent. Both p42 and p44 were found in the nucleus after stimulation, although a basal level of unphosphorylated protein was present before stimulation. The observed tyrosine phosphorylation of p42 and p44 was inhibited by genistein, a tyrosine kinase inhibitor, and tyrphostin 23, an epidermal growth factor receptor tyrosine kinase inhibitor. We conclude that MAPK is tyrosine phosphorylated in response to TGF-alpha stimulation of IEC-6 cells.

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ARG tyrosine kinase activity is inhibited by STI571

Blood ◽

10.1182/blood.v97.8.2440 ◽

2001 ◽

Vol 97 (8) ◽

pp. 2440-2448 ◽

Cited By ~ 173

Author(s):

Keiko Okuda ◽

Ellen Weisberg ◽

D. Gary Gilliland ◽

James D. Griffin

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Kinase Inhibitor ◽

Growth Factor Receptor ◽

Kinase Domain ◽

Fusion Partner ◽

Induced Apoptosis

Abstract The tyrosine kinase inhibitor STI571 inhibits BCR/ABL and induces hematologic remission in most patients with chronic myeloid leukemia. In addition to BCR/ABL, STI571 also inhibits v-Abl, TEL/ABL, the native platelet-derived growth factor (PDGF)β receptor, and c-KIT, but it does not inhibit SRC family kinases, c-FMS, FLT3, the epidermal growth factor receptor, or multiple other tyrosine kinases. ARG is a widely expressed tyrosine kinase that shares substantial sequence identity with c-ABL in the kinase domain and cooperates with ABL to regulate neurulation in the developing mouse embryo. As described here, ARG has recently been implicated in the pathogenesis of leukemia as a fusion partner of TEL. A TEL/ARG fusion was constructed to determine whether ARG can be inhibited by STI571. When expressed in the factor-dependent murine hematopoietic cell line Ba/F3, the TEL/ARG protein was heavily phosphorylated on tyrosine, increased tyrosine phosphorylation of multiple cellular proteins, and induced factor-independent proliferation. The effects of STI571 on Ba/F3 cells transformed with BCR/ABL, TEL/ABL, TEL/PDGFβR, or TEL/ARG were then compared. STI571 inhibited tyrosine phosphorylation and cell growth of Ba/F3 cells expressing BCR/ABL, TEL/ABL, TEL/PDGFβR, and TEL/ARG with an IC50 of approximately 0.5 μM in each case, but it had no effect on untransformed Ba/F3 cells growing in IL-3 or on Ba/F3 cells transformed by TEL/JAK2. Culture of TEL/ARG-transfected Ba/F3 cells with IL-3 completely prevented STI571-induced apoptosis in these cells, similar to what has been observed with BCR/ABL- or TEL/ABL-transformed cells. These results indicate that ARG is a target of the small molecule, tyrosine kinase inhibitor STI571.

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