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. 2012 Oct 30;109(44):18072-7.
doi: 10.1073/pnas.1209828109. Epub 2012 Oct 15.

Serine phosphorylation by SYK is critical for nuclear localization and transcription factor function of Ikaros

Fatih M Uckun  1 , Hong Ma, Jian Zhang, Zahide Ozer, Sinisa Dovat, Cheney Mao, Rita Ishkhanian, Patricia Goodman, Sanjive Qazi
Affiliations

Serine phosphorylation by SYK is critical for nuclear localization and transcription factor function of Ikaros

Fatih M Uckun et al. Proc Natl Acad Sci U S A. .

Abstract

Ikaros is a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis through transcriptional regulation of the earliest stages of lymphocyte ontogeny and differentiation. Functional deficiency of Ikaros has been implicated in the pathogenesis of acute lymphoblastic leukemia, the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros activity is considered of paramount importance, but the operative molecular mechanisms responsible for its regulation remain largely unknown. Here we provide multifaceted genetic and biochemical evidence for a previously unknown function of spleen tyrosine kinase (SYK) as a partner and posttranslational regulator of Ikaros. We demonstrate that SYK phoshorylates Ikaros at unique C-terminal serine phosphorylation sites S358 and S361, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Mechanistically, we establish that SYK-induced Ikaros activation is essential for its nuclear localization and optimal transcription factor function.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Serine phosphorylation and activation of recombinant IK by recombinant SYK. (A) After a cold kinase reaction, maltose-binding protein (MBP)-tagged recombinant IK was immunoprecipitated from the kinase reaction mixture and subjected to α-PS Western blot analysis. (B) Anti-IK Western blots of the samples shown in A. (C) Recombinant SYK showed autophosphorylation and it also phosphorylated MBP-IK1 in hot kinase assays. (D) Phospho amino acid analysis of the SYK-phosphorylated MBP-IK1 band showed S-phosphorylation. (E) MS analysis on trypsin-digested recombinant IK after an in vitro kinase reaction with purified recombinant SYK. (F) EMSAs were performed using increasing amounts (50, 100, 150, 200 ng per sample) of purified MBP-tagged recombinant IK1 protein that has been phosphorylated by recombinant SYK, C. (lane 1) IK-BS1 probe only. (lanes 2–5) Increasing amounts of SYK-phosphorylated MBP-IK1 were used in EMSA. (lane 6) MBP-IK1Phos(SYK) (200 ng) was mixed with 1 ng radiolabeled IK-BS1 in the presence of 100-fold excess unlabeled (cold) IK-BS1 for homologous competition. (lanes 7–10) Increasing amounts of unphosphorylated MBP-IK1 were used in EMSA. (lane 11) MBP-IK1 (200 ng) was mixed with 1 ng radiolabeled IK-BS1 in the presence of 100-fold excess of unlabeled (cold) IK-BS1.
Fig. 2.
Fig. 2.
Effects of siRNA-mediated depletion of native SYK on nuclear localization, sequence-specific DNA binding activity, and transcription factor function of native IK in human cells. (A) SYK vs. IK Western blot analysis of whole-cell lysates from 293T cells treated with medium only (CON), SYK siRNA, or BTK siRNA that was used as a control. (B) Confocal images of 293T cells stained with IK mAb and the blue fluorescent DNA dye DAPI following 72-h treatment with SYK siRNA or scrambled (scr) siRNA (included as a control). CON: No treatment. (Magnification: 630×.) (C) EMSAs measuring IK activity of nuclear extracts (NE) from untreated control (CON) 293T cells as well as 293T cells treated for 72 h with SYK siRNA, or scr-siRNA. (D) RT-PCR results for five randomly selected IK target genes after 72-h treatment with medium alone (CON), scrambled siRNA (sc-siRNA), IK siRNA, vs. SYK siRNA.
Fig. 3.
Fig. 3.
Functional studies on mutant IK proteins generated by site-directed mutagenesis. (A–C) Confocal images of 293T cells expressing the mutant IK proteins following treatment with SYK siRNA. (Magnification: 630×.) (D) EMSAs measuring IK activity of nuclear extracts (NE) from 293T cells transfected with expression vectors for wild-type or mutant IK proteins. (E) RT-PCR results for three randomly selected Ikaros target genes in 293T cells expressing wild-type or mutant IK proteins.
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