Abstract
4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine (PP1) was identified as an Src-selective tyrosine kinase inhibitor and has been used extensively to investigate signaling pathways involving Src kinases, including events downstream of the stem cell factor (SCF) receptor c-Kit. While investigating the role of Src kinases in SCF signaling, we found that PP1 completely abrogated the proliferation of M07e cells in response to SCF. PP1 inhibited SCF-induced c-Kit autophosphorylation in intact cells and blocked the activation of mitogen-activated protein kinase and Akt. In vitro kinase assays using immunoprecipitated c-Kit confirmed direct inhibition by PP1. SCF-induced c-Kit phosphorylation was also inhibited by the related inhibitor 4-amino-5- (4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP2) and by STI571 but not by the Src inhibitor SU6656. PP1 inhibited the activity of mutant constitutively active forms of c-Kit (D814V and D814Y) found in mast cell disorders, and triggered apoptosis in the rat basophilic leukemia cell line RBL-2H3 that expresses mutant c-Kit. In addition, PP1 (and PP2) inhibited the in vitro kinase activity and autophosphorylation in whole cells of p210 Bcr-Abl. PP1 reduced the constitutive activation of signal transducer and activators of transcription 5 and mitogen-activated protein kinase and triggered apoptosis in FDCP1 cells expressing Bcr-Abl. These results have implications for the use of PP1 in investigating intracellular signaling and suggest that PP1 or related compounds may be useful in the treatment of malignant diseases associated with dysregulated c-Kit or Abl tyrosine kinase activity.
Highlights
4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) was identified as an Src-selective tyrosine kinase inhibitor and has been used extensively to investigate signaling pathways involving Src kinases, including events downstream of the stem cell factor (SCF) receptor c-Kit
It has previously been shown that of the Src family of tyrosine kinases, Lyn is present in M07e cells and that activation of this kinase can be detected after SCF stimulation [3, 21]
PP1 was originally described as a selective, ATP-competitive inhibitor of Src family kinases [1] and has been widely used to investigate the contribution of Src kinases to a number of biological functions
Summary
4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) was identified as an Src-selective tyrosine kinase inhibitor and has been used extensively to investigate signaling pathways involving Src kinases, including events downstream of the stem cell factor (SCF) receptor c-Kit. 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. PP1 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
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