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. 2012 Jan 20;417(3):1058-62.
doi: 10.1016/j.bbrc.2011.12.095. Epub 2011 Dec 27.

Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

Srividya Venkitachalam  1 , Fu-Yu Chueh, Chao-Lan Yu
Affiliations

Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

Srividya Venkitachalam et al. Biochem Biophys Res Commun. .

Abstract

LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

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Figures

Fig. 1
Fig. 1
Elevated Lmo2 expression in Lck-transformed cells. (A) Total RNA was isolated from mouse BYDP and LSTRA T cells, reverse transcribed, and then subjected to real-time PCR analysis using primers specific for mouse Lmo2 and actin mRNAs. Data from triplicates were normalized to actin and expressed as fold change to BYDP cells. (B) T-REx-293/Y505FLck cells were either left untreated (-) or treated (+) with doxycycline (Dox) to induce the expression of Y505FLck. RNA was isolated and real-time PCR was performed using primers specific for human Lmo2 and actin mRNAs. Data from triplicates were normalized to actin and expressed as fold change to cells that were not treated with doxycycline. Statistical analyses show the results from three independent studies, ** p<0.01.
Fig. 2
Fig. 2
Nuclear localization of oncogenic Lck kinase. (A) Cytosolic (Cyt) and nuclear (Nuc) fractions were isolated from LSTRA cells (lanes 1-2) as well as T-REx-293/Y505FLck cells either untreated or treated with doxycycline (lanes 3-6). Normalized total proteins were analyzed by immunoblotting with antibodies specific for Lck, GAPDH and Lamin B1. (B) LSTRA and doxycycline-treated T-REx-293/Y505FLck (293Lck) cells were stained for Lck (red) and nuclei were visualized by DAPI staining (blue). Magnification is 200X for LSTRA and 60X for HEK 293 cells.
Fig. 3
Fig. 3
Phosphorylation of positive regulatory tyrosine of nuclear Lck. Cytosolic and nuclear fractions were isolated from LSTRA (A) and T-REx-293/Y505FLck (B) cells as described for Fig. 2. Normalized total proteins were immunoprecipitated with anti-Lck antibody, followed by immunoblotting with antibody specific for Tyr 394-phosphorylated Lck. The membrane was stripped and then reblotted for total Lck using anti-Lck (A) or anti-Myc tag (B) antibody. The arrowheads mark the positions of immunoglobulin heavy chain (IgH).
Fig. 4
Fig. 4
The effect of Lck on histone H3 phosphorylation and Lmo2 gene promoter. (A) Whole cell lysates were prepared from BYDP, LSTRA and T-REx-293/Y505FLck without or with doxycycline treatment. Normalized total proteins were subjected to sequential immunoblotting using antibodies specific for Tyr 41-phosphorylated histone H3 and total histone H3. (B) Doxycycline-treated T-REx-293/Y505FLck cells were subjected to chromatin immunoprecipitation with control IgG or anti-Myc tag antibody, followed by real-time PCR using primers specific for the promoter region of human Lmo2. Data from triplicates are presented as fold enrichment in Lck binding to the Lmo2 promoter region. ** p<0.01.
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References

    1. McCormack MP, Rabbitts TH. Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 2004;350:913–922. - PubMed
    1. Nam CH, Rabbitts TH. The role of LMO2 in development and in T cell leukemia after chromosomal translocation or retroviral insertion. Mol. Ther. 2006;13:15–25. - PubMed
    1. Oram SH, Thoms JA, Pridans C, Janes ME, Kinston SJ, Anand S, Landry JR, Lock RB, Jayaraman PS, Huntly BJ, Pimanda JE, Gottgens B. A previously unrecognized promoter of LMO2 forms part of a transcriptional regulatory circuit mediating LMO2 expression in a subset of T-acute lymphoblastic leukaemia patients. Oncogene. 2010;29:5796–5808. - PubMed
    1. Rabbitts TH, Axelson H, Forster A, Grutz G, Lavenir I, Larson R, Osada H, Valge-Archer V, Wadman I, Warren A. Chromosomal translocations and leukaemia: a role for LMO2 in T cell acute leukaemia, in transcription and in erythropoiesis. Leukemia. 1997;11(Suppl 3):271–272. - PubMed
    1. Landry JR, Kinston S, Knezevic K, Donaldson IJ, Green AR, Gottgens B. Fli1, Elf1, and Ets1 regulate the proximal promoter of the LMO2 gene in endothelial cells. Blood. 2005;106:2680–2687. - PubMed

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