The Src-selective Kinase Inhibitor PP1 Also Inhibits Kit and Bcr-Abl Tyrosine Kinases

Abstract

4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1) was identified as anThe pyrazolo-pyrimidine compound PP11 was identified as a high potency inhibitor of Src tyrosine kinase family members that acts as a competitive inhibitor of ATP binding (1). PP1 has been used in a number of studies to evaluate the role of Src tyrosine kinases in cellular function (2-7). PP1 does not affect the activity of other non-receptor tyrosine kinases, such as Jak-2 and Zap-70 (1). Src kinases have been reported to be critically involved in signaling via the stem cell factor (SCF) receptor c-Kit; inhibition of Src kinases has been shown to abrogate SCF induced proliferation of hemopoietic (3) and small cell lung cancer cells (8) and to block internalization of c-Kit after ligand binding (4). These findings have relied largely on the use of PP1 as a Src-selective inhibitor. c-Kit is expressed on hemopoietic progenitors, mast cells, and germ cells (for review, see Refs. 9 and 10). Loss of either c-Kit or its ligand in mice results in impaired hemopoiesis (in particular anemia), abnormalities of mast cell development, and impaired melanogenesis and gametogenesis (11). c-Kit has been implicated in tumor cell development, including mast cell disorders, (12) acute myeloid leukemia, gastrointestinal stromal tumors (13), Ewing's sarcoma, peripheral neuro-ectodermal tumors, malignant melanoma (11) and cancers of lung, ovary, and breast (14 -16). In some instances, this is because of activating mutations in c-Kit (e.g. in mastocytosis, acute myeloid leukemia, and gastrointestinal stromal tumors) (17); in others, the receptor is wild type but there is associated autocrine production of SCF (16).The development of inhibitors of c-Kit-mediated signals would be useful to investigate the role of c-Kit in maintaining these tumors and could provide the basis for a potential therapeutic agent. In this study, we have examined the effects of PP1 on signaling via c-Kit. We find that PP1 and the related compound PP2 block SCF-induced proliferation and activation of the ERK mitogen-activated protein kinase and Akt. This is associated with an inhibition of c-Kit autophosphorylation both in intact cells and in in vitro kinase assays. PP1 decreases the activity of constitutively active c-Kit and triggers the apoptosis of mast cell leukemia cells expressing this mutant. PP1 also inhibits Bcr-Abl kinase activity and triggers apoptosis in BcrAbl-dependent cells. These results have implications for the use of PP1 and related compounds in the experimental investigation of cell signaling pathways and suggest that this family of molecules may be useful in the development of treatment for diseases with abnormal c-Kit signaling. MATERIALS AND METHODSCell Culture-The RBL-2H3 cell line (18) (kindly supplied by S. Cockcroft,

Collagen type I selectively activates ectodomain shedding of the discoidin domain receptor 1: Involvement of Src tyrosine kinase

“…5C). The relatively larger inhibitory effects of PP1 compared to SrcRF might reflect direct inhibition of the intrinsic kinase activity of DDR1, since PP1 has been shown to affect a number of kinases in addition to Src [Tatton et al, 2003;Bain et al, 2003]. Nevertheless, when considered in conjunction with the effect of dominant negative Src, the results suggest that autophosphorylation of DDR1 and activation of a Src-family kinase are both required for maximal phosphorylation and cleavage of DDR1 in response to collagen.…”

The Src tyrosine kinase Hck is required for Tel-Abl- but not for Tel-Jak2-induced cell transformation

“…Interestingly, this interaction does not require the phosphorylation of the partners (Klejman et al, 2002) and our unpublished data also argue for a constitutive Stat5/Hck interaction in Ba/F3 cells expressing Tel-Abl that does not require the phosphorylation of Stat5 or Hck. However, since treatment with imatinib mesylate or PP2 which has been reported to inhibit also Bcr-Abl (Tatton et al, 2003;Warmuth et al, 2003) and herein Tel-Abl, suppresses Stat5 phosphorylation, Tel-Abl itself or another Src kinase may activate these factors. Although the Lyn kinase was shown to physically interact with and activate Stat5 factors (Danhauser-Riedl et al, 1996;Chin et al, 1998;Tilbrook et al, 2001), the absence of any effect of the dominant-negative form of the kinase on Stat5 phosphorylation in Tel-Abl-expressing cells do not argue for a role of Lyn in this process (data not shown).…”

Gain-of-Function Mutations in the Extracellular Domain of KIT Are Common in Canine Mast Cell Tumors

“…Fourth, the inhibitors could affect multiple enzymes or pathways. Indeed, AG1296 and PP2 are known to inhibit several tyrosine kinases (30)(31)(32), and it is possible that the phosphorylation of downstream effectors or the activity of tyrosine kinases downstream of KIT is more sensitive to AG1296 and PP2 than KIT itself. Because both AG1296 and PP2 were potent inhibitors of mutant KIT-induced cell proliferation in all cases, it seems that, at a minimum, tyrosine kinase activity is necessary for their transforming activity.…”