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. 2015 Jul;34(1):43-50.
doi: 10.3892/or.2015.3990. Epub 2015 May 19.

Nuclear lymphocyte-specific protein tyrosine kinase and its interaction with CR6-interacting factor 1 promote the survival of human leukemic T cells

Shahrooz Vahedi  1 , Fu-Yu Chueh  1 , Sujoy Dutta  1 , Bala Chandran  1 , Chao-Lan Yu  1
Affiliations

Nuclear lymphocyte-specific protein tyrosine kinase and its interaction with CR6-interacting factor 1 promote the survival of human leukemic T cells

Shahrooz Vahedi et al. Oncol Rep. 2015 Jul.

Abstract

Overexpression and hyperactivation of lymphocyte-specific protein tyrosine kinase (Lck) have been associated with leukemia development. We previously showed that, other than its known function as a cytoplasmic signal transducer, Lck also acts as a nuclear transcription factor in mouse leukemic cells. In the present study, we demonstrated the presence of nuclear Lck in human leukemic T cells and in primary cells. We further established a positive correlation between Lck nuclear localization and its kinase activity. Proteomic analysis identified CR6-interacting factor 1 (CRIF1) as one of the Lck-interacting proteins. CRIF1 and Lck association in the nucleus was confirmed both by immunofluorescence microscopy and co-immunoprecipitation in human leukemic T cells. Close-range interaction between Lck and CRIF1 was validated by in situ proximity ligation assay (PLA). Consistent with the role of nuclear CRIF1 as a tumor suppressor, CRIF1 silencing promotes leukemic T cell survival in the absence of growth factors. This protective effect can be recapitulated by endogenous Lck or reconstituted Lck in leukemic T cells. All together, our results support a novel function of nuclear Lck in promoting human leukemic T cell survival through interaction with a tumor suppressor. It has important implications in defining a paradigm shift of non-canonical protein tyrosine kinase signaling.

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Figures

Figure 1
Figure 1
Nuclear localization of endogenous Lck in T cell lines and primary cells. (A) Nuclear (Nuc) fractions isolated from Jurkat and LSTRA cell lines were analyzed by Lck immunoblotting. GAPDH (cytosolic marker) and lamin B1 (nuclear marker) immunoblotting were also performed to verify the purity of the nuclear fraction. Jurkat whole cell lysate (WCL) was used as a positive control for markers (lane 3). (B) Jurkat cells were subjected to immunofluorescence microscopy using an anti-Lck antibody (red). Nuclei were counterstained with DAPI (blue). Nuclear staining of Lck is indicated by an arrow in the enlarged merged image. Scale bars of 5 μm are shown at the bottom of the microscopy images. (C) Mouse splenocytes stimulated with pervanadate for 5 min (+PV) or left unstimulated (−PV) were analyzed by anti-Lck immunofluorescence microscopy (green) with DAPI counterstain (blue). Lck, lymphocyte-specific protein tyrosine kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 2
Figure 2
Contribution of exogenous Lck kinase activity in its nuclear translocation. (A) Nuclear fractions were isolated from HEK293T cells transfected with wild-type (WT) and three different mutant Lck constructs (lanes 1–4). Equal amount of proteins were analyzed by Lck immunoblotting (top panel). Whole cell lysates (WCL) prepared from 10% of the transfected cells were included to determine the expression levels of exogenous Lck proteins (lanes 5–8, top panel). Fraction purity was verified by GAPDH and lamin B1 immunoblotting. (B) Expression levels of nuclear Lck and total Lck were quantitated in transfected HEK293T cells to obtain the ratios of nuclear expression. For each set of transfection experiments, the ratio of nuclear wild-type Lck was set as 100%. Statistical analysis was performed on three independent transfection experiments, *P<0.05, **P<0.01, ***P<0.001. Lck, lymphocyte-specific protein tyrosine kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 3
Figure 3
Contribution of endogenous Lck kinase activity in its nuclear translocation. (A) Nuclear and cytosolic fractions were isolated from Jurkat and Jcam cells either unstimulated (Un) or stimulated with pervanadate for 5 min (S). Equal amount of proteins from each set of lysates was immunoprecipitated (IP) with an anti-Lck antibody, and then subjected to immunoblotting with an anti-phospho-Src family (Y416) antibody to visualize Lck phosphorylation (pLck). The amount of total Lck was determined by sequential blotting with the anti-Lck antibody. A fraction of the total lysates was probed for GAPDH and lamin B1 to verify fraction purity. (B) Jurkat cells were treated with dasatinib or vehicle control for 2 h. Whole cell lysates (WCL) were prepared from a fraction of the cells and analyzed by immunoblotting using antibodies specific for phospho-Y416-Src family (pSrc) and GAPDH (lanes 1 and 2). Nuclear proteins isolated from the remaining cells were subjected to immunoblotting using antibodies specific for Lck, lamin B1 and Eps15 (cytosolic marker) (lanes 5 and 6). Equal amount of whole cell lysates was also analyzed by immunoblotting using antibodies specific for Lck, lamin B1 and Eps15 (lanes 3 and 4). Lck, lymphocyte-specific protein tyrosine kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Eps15, epidermal growth factor receptor substrate 15.
Figure 4
Figure 4
Lck interacts with CRIF1. (A) Jurkat cells were subjected to immunofluorescence microscopy with two-color staining for CRIF1 (green) and Lck (red). Cells were also counterstained with DAPI to visualize nuclei (blue). White arrows in the enlarged three-color merged image indicate co-localization of Lck and CRIF1 in the nucleus (white dots). (B) Nuclear proteins isolated from Jurkat cells were immunoprecipitated with either an anti-CRIF1 antibody (lane 2) or control IgG (lane 3), and then subjected to Lck and CRIF1 immunoblotting. A fraction of Jurkat whole cell lysate was loaded as positive controls (lane 1). (C) Jurkat and Jcam cells were subjected to PLA microscopy using primary antibodies specific for Lck and CRIF1. The areas bordered by white lines are enlarged on the right for enhanced visualization. Red fluorescence indicates Lck and CRIF1 interaction in situ. White arrows denote Lck and CRIF1 interaction in the nucleus. Lck, lymphocyte-specific protein tyrosine kinase; CRIF1, CR6-interacting factor 1; PLA, proximity ligation assay.
Figure 5
Figure 5
CRIF1 knockdown and association with Lck confer resistance to cell death induced by serum deprivation. (A) Whole cell lysates prepared from Jcam cells stably transduced with sh-control and sh-CRIF1 (CRIF1 KD) were subjected to CRIF1 and GAPDH immunoblotting. (B) Cell death after serum deprivation was measured in transduced Jcam cells and presented as a percentage in comparison to the sh-control Jcam cells. (C) Whole cell lysates were prepared from Jurkat, Jacm, and Jcam reconstituted with wild-type Lck (Jcam/Lck). Normalized proteins were immunoprecipitated with an anti-CRIF1 antibody (lanes 2–4) or control IgG (lane 1), and then subjected to sequential blotting with anti-Lck and anti-CRIF1 antibodies. (D) Cell death after serum deprivation was measured in Jurkat, Jcam and Lck-expressing Jcam cells by trypan blue exclusion assay. The ratio of cell death in the Jcam cells was set as 100% in each set of experiment. Statistical analysis was performed using data from three independent experiments, *P<0.05 or non-significant (n.s.). Lck, lymphocyte-specific protein tyrosine kinase; CRIF1, CR6-interacting factor 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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