Tomoyuki Shishido

Identification of B cell adaptor for PI3-kinase (BCAP) as an Abl interactor 1-regulated substrate of Abl kinases.

In previous work we showed that Abl interactor 1 (Abi‐1), by linking enzyme and substrate, promotes the phosphorylation of Mammalian Enabled (Mena) by c‐Abl. To determine whether this mechanism extends to other c‐Abl substrates, we used the yeast two‐hybrid system to search for proteins that interact with Abi‐1. By screening a human leukocyte cDNA library, we identified BCAP (B‐cell adaptor for phosphoinositide 3‐kinase) as another Abi‐1‐interacting protein. Binding experiments revealed that the SH3 domain of Abi‐1 and the C‐terminal polyproline structure of BCAP are involved in interactions between the two. In cultured cells, Abi‐1 promoted phosphorylation of BCAP not only by c‐Abl but also by v‐Abl. The phosphorylation sites of BCAP by c‐Abl were mapped to five tyrosine residues in the C‐terminal region that are well conserved in mammals. These results show that Abi‐1 promotes Abl‐mediated BCAP phosphorylation and suggest that Abi‐1 in general coordinates kinase‐substrate interactions.

Abl kinase interacts with and phosphorylates vinexin

Non‐receptor tyrosine kinase Abl is a well known regulator of the actin‐cytoskeleton, including the formation of stress fibers and membrane ruffles. Vinexin is an adapter protein consisting of three SH3 domains, and involved in signal transduction and the reorganization of actin cytoskeleton. In this study, we found that vinexin α as well as β interacts with c‐Abl mainly through the third SH3 domain, and that vinexin and c‐Abl were colocalized at membrane ruffles in rat astrocytes. This interaction was reduced by latrunculin B, suggesting an F‐actin‐mediated regulatory mechanism. We also found that vinexin α but not β was phosphorylated at tyrosine residue when c‐Abl or v‐Abl was co‐expressed. A mutational analysis identified tyrosine 127 on vinexin α as a major site of phosphorylation by c‐ or v‐Abl. These results suggest that vinexin α is a novel substrate for Abl.

NESH (Abi-3) is present in the Abi/WAVE complex but does not promote c-Abl-mediated phosphorylation

Abl interactor (Abi) was identified as an Abl tyrosine kinase‐binding protein and subsequently shown to be a component of the macromolecular Abi/WAVE complex, which is a key regulator of Rac‐dependent actin polymerization. Previous studies showed that Abi‐1 promotes c‐Abl‐mediated phosphorylation of Mammalian Enabled (Mena) and WAVE2. In addition to Abi‐1, mammals possess Abi‐2 and NESH (Abi‐3). In this study, we compared the three Abi proteins in terms of the promotion of c‐Abl‐mediated phosphorylation and the formation of Abi/WAVE complex. Although Abi‐2, like Abi‐1, promoted the c‐Abl‐mediated phosphorylation of Mena and WAVE2, NESH (Abi‐3) had no such effect. This difference was likely due to their binding abilities as to c‐Abl. Immunoprecipitation revealed that NESH (Abi‐3) is present in the Abi/WAVE complex. Our results suggest that NESH (Abi‐3), like Abi‐1 and Abi‐2, is a component of the Abi/WAVE complex, but likely plays a different role in the regulation of c‐Abl.

CrkII induces serum response factor activation and cellular transformation through its function in Rho activation

CrkII belongs to the adaptor protein family that plays a crucial role in signal transduction. In order to better understand the biological functions of CrkII, we focused on the regulation of gene expression by CrkII. Various transcriptional control elements were examined for their activation by CrkII-expression, and we found that CrkII selectively activates the serum response element (SRE), a transcriptional control element of immediate-early genes. This SRE activation induced by CrkII-overexpression was mediated by the serum response factor (SRF) via Rho. Indeed, we confirmed that the amount of activated Rho was increased in the CrkII-expressing cells. Moreover, we showed that when overexpressed, CrkII induces the cellular transformation of NIH 3T3 cells and that a dominant negative mutant of Rho suppresses this transformation, strongly suggesting that activation of Rho is essential for the transforming activity by CrkII. Furthermore, we also found that CrkII and Gα12, a member of the heterotrimeric G proteins, synergistically activates Rho as well as the SRF, and that an SH3 mutant of CrkII can inhibit the Gα12-induced activation of SRF. These results strongly suggest that CrkII is involved in the activation of Rho and SRF by Gα12. Our study provides strong evidence that Rho activation plays a crucial role in CrkII-mediated signals to induce gene expression and cellular transformation.

Loss of c-abl facilitates anchorage-independent growth of p53- and RB- deficient primary mouse embryonic fibroblasts

The c-abl tyrosine kinase is the proto-oncogene of the v-abl oncogene of the Abelson murine leukemia virus. Although mutational variants of c-Abl can exhibit gain of function and can produce a transformed phenotype, the function of c-Abl in transformation remained unclear. Here, we report that the loss of c-abl facilitates transformation. c-abl-knockout mouse embryonic fibroblasts (MEFs) immortalized by SV40 T antigen acquired anchorage-independent growth, and by constructing mutational variants of T antigen we showed that binding of large T antigen to p53 and RB was necessary to induce anchorage-independent growth. Although c-abl/p53 double-knockout MEFs did not undergo anchorage-independent growth, those expressing human papilloma virus 16 E7, which mainly inactivates RB, did. Our results show that the loss of c-abl facilitates anchorage-independent growth in the context of p53 and RB deficiency, and suggest that loss of function of c-abl facilitates some types of transformation.

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 μM for EC50 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.