Imatinib mesylate (STI571)-induced cell edge translocation of kinase-active and kinase-defective Abelson kinase: requirements of myristoylation and src homology 3 domain.
4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide methanesulfonate (STI571) is the first successful target-based drug with excellent potency against chronic myelogenous leukemia. Studies on this compound have illuminated potentials and problems of kinase inhibitors in the treatment of cancer. As found in crystal structures, STI571-bound Abelson kinase (abl) is believed to form closed conformation with N-terminal regulatory domains. Here we present evidence of distinct STI571-induced modulation of abl functions using high-resolution live-cell imaging approaches. Within lamellipodia of fibroblast cells, STI571 was found to induce rapid translocation of abl to the lamellipodium tip. Quantitative analysis yielded 0.81 and 1.8 microM for EC(50) values of STI571-induced cell edge translocation of abl-KD-green fluorescent protein (GFP) and wild-type abl-GFP, respectively. It also revealed adverse response of drug-resistant abl-T334I to STI571, suggesting that drug binding to abl-GFP triggers translocation. N-myristoylation and the src homology 3 (SH3) domain were required for this translocation, whereas disruption of intramolecular interactions of these motifs enhanced cell-edge association of abl. An intact C-terminal last exon region in abl, but not its F-actin binding, was required for efficient cell-edge translocation. Moreover, single-molecule observation revealed an STI571-induced rapid increase in slow diffusive species of abl in both the tip and the body region of lamellipodia. These results suggest that although activated abl translocates to the cell edge at its open state, STI571 can also bind and lock abl in the open and membrane-tethered conformation as long as the SH3 domain and the C-terminal region are intact. High-resolution imaging can be a powerful tool for elucidating inhibitor modulation of abl functions under intracellular environment.
Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan.
Akiko
A
Fujita
Tomoyuki
T
Shishido
Yunfeng
Y
Yuan
Eiji
E
Inamoto
Shuh
S
Narumiya
Naoki
N
Watanabe
D000818:Animals; D001549:Benzamides; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D000068877:Imatinib Mesylate; D051379:Mice; D019814:Myristic Acid / Q000378:metabolism*; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology*; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics*; D011743:Pyrimidines / Q000494:pharmacology*; D014162:Transfection; D014982:Xenopus laevis / Q000378:metabolism; D018909:src Homology Domains / Q000235:genetics*
D001549:Benzamides; D016895:Culture Media, Serum-Free; D005456:Fluorescent Dyes; D010879:Piperazines; D011743:Pyrimidines; D019814:Myristic Acid; D049452:Green Fluorescent Proteins; D000068877:Imatinib Mesylate; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; D016315:Proto-Oncogene Proteins c-abl