Biochemical and Biophysical Research Communications
Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39
Introduction
RBM39 (also called HCC1.4 or CAPERα) was first identified as an auto-antigen in a hepatocellular carcinoma patient [1], [2] and as an inhibitor of v-Rel-mediated lymphocyte transformation [3]. RBM39, which was also shown to be a transcriptional co-activator of activating protein-1 (AP-1) and the estrogen receptor ERα, is involved in the regulation of the transcriptional responses of these proteins and promotes transcriptional initiation [1], [4]. As a highly homologous protein to U2AF65, which is an essential serine-/arginine-rich (SR-rich) splicing factor, RBM39 contains a long arginine- and serine-rich domain (RS domain) and three RNA-recognition motifs (RRMs) and shares notable sequence similarity with U2AF65 but lacks a U2AF ligand motif [5]. Immunofluorescence microscopy has shown that RBM39 is located in a speckled network in the nucleus and co-localizes with splicing factor SC35 and uridine-rich small nuclear RNAs. RBM39 may play a role in hormone-dependent transcriptional activation and function as a splicing cofactor for steroid receptors [4], [6], [7].
The mammalian Abl nonreceptor tyrosine kinase (c-Abl), a modular protein that is widely expressed in adult and fetal tissues, has multiple functions [8], [9]. The N-terminal portion of c-Abl is composed of Src homology (SH) 2, SH3, and kinase domains [10], [11]. The existence of C-terminal DNA binding motifs and nuclear localization signals in c-Abl enables the protein to shuttle between the cytoplasmic and nuclear compartments [12], [13], increasing its exposure to additional Abl kinase substrates. c-Abl is important for multiple cellular processes, including cell proliferation, apoptosis, differentiation, survival, adhesion, and migration, as well as inflammation and stress responses [8], [9], [14]. It is well established that the effects of c-Abl are mediated by protein–protein interactions and the phosphorylation of substrate proteins by tyrosine kinase activity. c-Abl functions in both proapoptotic and antiapoptotic pathways, particularly under conditions of irradiation, DNA damage and other stressors, such as reactive oxygen species (ROS) [8], [9], [14], [15], [16].
Although the phenomenon that steroid hormone receptors recruit coregulators such as RBM39 to modulate their transcriptional response and facilitate transcriptional initiation has been well documented, the regulation of RBM39 by tyrosine kinases is largely unknown. In this report, we showed that c-Abl interacts with and phosphorylates RBM39. In addition, the transcriptional coactivation activity of RBM39 for ERα and PRβ was promoted by c-Abl.
Section snippets
Vectors
Flag-tagged vectors expressing RBM39, RBM39(Y95F), RBM39(Y99F), RBM39(Y95FY99F), RBM39(Y95FY99FY475F) and RBM39(Y95FY99FY475FY505F) were constructed by cloning the genes into the pcDNA3-based Flag vector (Thermo Fisher, CA, USA). The Myc-tagged c-Abl and c-Abl(K290R) plasmids were constructed by cloning these genes into the pCMV-Myc vector (BD Biosciences Clontech, CA, USA). Flag-ERα and Flag-PRβ were kindly provided by Drs. Qinong Ye and Xuemin Zhang from the Academy of Military Medical
c-Abl interacts with RBM39
Using yeast-two hybrid analysis, we previously noted that c-Abl interacts with RBM39 (data not shown). To substantiate the interaction between c-Abl and RBM39, lysates from wild-type MEFs were immunoprecipitated with the anti-RBM39 antibody and were probed for anti-c-Abl and anti-RBM39. Immunoblotting showed that c-Abl was present in the anti-RBM39 immunoprecipitate but was not pulled down with the rabbit IgG antibody in MEFs (Fig. 1A). To confirm the interaction between c-Abl and RBM39, 293T
Discussion
RBM39 was previously identified as an auto-antigen in a hepatocellular carcinoma patient [1], [2] and as an inhibitor of v-Rel-mediated lymphocyte transformation [3]. RBM39 was also shown to be a transcriptional co-activator of AP-1 and ERα [1], [4]. A highly homologous protein to U2AF65, which is an essential SR-rich splicing factor, RBM39 contains a long RS domain and three RRMs. RBM39 shares notable sequence similarity with U2AF65 but lacks a U2AF ligand motif [5]. The functions of many
Acknowledgments
This work was supported by the National Natural Science Foundation of China [30871240, 31070674]. We thank American Journal Experts (AJE) for language help.
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