Amino terminal tyrosine phosphorylation of human MIXL1
© Guo and Nagarajan. 2006
Received: 13 April 2006
Accepted: 05 December 2006
Published: 05 December 2006
Seven members of the Mix family of paired-type homeoproteins regulate mesoderm/endoderm differentiation in amphibians. In mammals, the MIXL1 (Mix. 1 homeobox [Xenopus laevis]-like gene 1) gene is the sole representative of this family. Unlike the amphibian Mix genes that encode an open reading frame of >300 amino acids, mammalian MIXL1 encodes a smaller protein (~230aa). However, mammalian MIXL1 contains a unique proline-rich domain (PRD) with a potential to interact with signal transducing Src homolgy 3 (SH3) domains. Notably, human MIXL1 also contains a unique tyrosine residue Tyr20 that is amino-terminal to the PRD. Here we report that mammalian MIXL1 protein is phosphorylated at Tyr20 and the phosphorylation is dramatically reduced in the absence of PRD. Our findings are consistent with Tyr20 phosphorylation of MIXL1 being a potential regulatory mechanism that governs its activity.
Mix. 1, a paired-like homeobox gene, was initially identified as an inducer of ventral mesoderm and/or endoderm in Xenopus [1, 2]. Subsequently, several closely related genes, Mix 2–4, Bix 1–4, and Mixer, were isolated and found to regulate mesoderm and/or endoderm formation [3–6]. However, in chicken (CMIX), mice (MIXL1/Mml) and humans, the Mix-like homeobox (MIXL1) genes appear to be single copies [7–12]. Additionally, mammalian MIXL1 encodes a smaller protein of ~230 amino acids, in contrast to the ~340 amino acid proteins encoded by the Xenopus genes. Nonetheless, almost all of the Mix family members are modular with a highly conserved paired-type homeodomain and a conserved carboxy-terminal acidic domain (CAD). A distinguishing feature of CMIX, Mml/MIXL1 and human MIXL1 is the presence of proline-rich domains (PRD). Both mouse and human MIXL1 contain an amino-terminal PRD between residues 31–60; in chicken however, the PRD appears to be carboxyl to the homeodomain raising the possibility that the function of this domain may be modular.
The Xenopus Mix/Bix genes are expressed in ventral mesoderm and/or endoderm [1, 3–6]. Similarly, the expression of mouse Mml/MIXL1 or chicken CMIX initially occurs in visceral endoderm and, becomes restricted to primitive streak and nascent mesoderm at gastrulation; in mice, this includes the hemangioblast, a precursor of hematopoietic and vascular stem cells [9–14]. The expression of human MIXL1 is restricted to progenitors and secondary lymph tissues in adults . The temporal and spatial expression pattern of Mix-like genes suggests that these genes are tightly regulated during embryonic development and hematopoietic differentiation. The MIX family appears to be regulated by at least three signaling pathways: TGFβ/Activin/BMP, FGF, and p53 [2–4, 6, 15–19].
A role for the Mix gene family in development is suggested by both gain-and loss-of-function experiments. Xenopus Mix.1 gene is implicated in the process of patterning ventral mesoderm to hematopoietic fate induced by BMP-4  and in endoderm development by synergizing with other regulatory molecules such as Siamois . Similar to Mix.1, ectopic expression of human MIXL1 induced embryonic hematopoiesis in Xenopus animal caps. Homozygous disruption of mouse Mml/MIXL1 resulted in a marked thickening of the primitive streak, severe defects in paraxial mesoderm, and absence of heart tube and gut . In vitro ES differentiation assays further demonstrated that murine Mml/MIXL1 to be BMP4 responsive and required for efficient hematopoiesis .
In contrast to the developmental studies on this gene family, nothing is known about biochemical pathways regulating mammalian MIXL1. Absence of multiple family members coupled with the gain of PRD in mammals, raises a number of mechanistic possibilities for similar cell fate or differentiation pathways regulated in amphibians and mammals. One of these may be tissue- or developmental stage-specific phosphorylation of MIXL1 that may mimic the diverse regulatory functions by multiple members in Xenopus. In this report, we show that mammalian MIXL1 protein is readily phosphorylated at the amino terminal tyr20. Tyr20 phosphorylation of MIXL1, a potential regulatory mechanism governing its activity is dramatically reduced in the absence of PRD.
Materials and methods
Plasmid construction and mutagenesis
The full-length human MIXL1 ORF, ΔCAD mutants with truncation of carboxy-terminal acidic domain and ΔN25 mutant with truncation of both amino-terminal 25 bp region and carboxy-terminal acidic domain were amplified by PCR and cloned into expression vectors CMV5 (a kind gift from David W. Russell, UTSW at Dallas, TX) and CMV2-flag (Sigma, St. Louis, MO) to generate CMV5-MIXL1, CMV2-Flag-MIXL1, CMV5-ΔCAD, CMV2-flag-ΔCAD and CMV2-flag-ΔN25. The amino terminal of CMV2-flag-ΔCAD was replaced with the 1–93 bp amino-terminal portion of MIXL1 to generate the construct CMV2-flag-ΔPC lacking both PRD and CAD domains. Y110F mutation was introduced by PCR with a primer containing an A-to-T (Tyr-to-Phe) point mutation. The MIXL1 carboxy-terminal portion (301–699) of the construct CMV2-flag – ΔCAD or CMV2-flag-ΔN25 was replaced with the amplified MIXL1 fragments carrying the A-to-T point mutation to generate the construct CMV2-flag-Y110F or CMV2-flag-Δ2Y. Accuracy of the generated constructs was confirmed by double stranded sequencing.
Cell culture and transfection
HEK 293T cells were grown in modified Eagle's medium (MEM, Invitrogen) supplemented with 10% FBS, 0.1 mM non-essential amino acids and 1 mM sodium pyruvate (Invitrogen) at 37°C in 10% CO2. For transfection, cells were plated at a density of 2 × 105 cells per well in a 6-well plate 2 days prior to transfection. The transfections were performed using LipofectAmine (Invitrogen) according to the manufacturer's protocols. Total amount of transfected DNA was adjusted to 1 μg per well by using appropriate parental vectors. Cells were harvested for nuclear extraction 48 hours after transfection. For immunoprecipitation experiments, the transfections were scaled up to 100 mm plates.
Immunoprecipitation and immunobloting
Nuclear extracts were prepared from transfected 293T cells  and diluted to approximately 1.0 μg protein/μL lysate. Briefly, 500 μL of the fresh nuclear extracts were pre-cleared with 50 μL of protein-A-agarose bead slurry (50% v/v, Roche, Indianapolis, IN) at 4°C for 30 minutes in an orbital shaker. After centrifugation at 14,000 × g at 4°C for 10 minutes, supernatant was mixed with 5 μg of the murine monoclonal antibody 4G10 (Upstate) or the isotypic control (murine monoclonal antibody against the V5 epitope-Invitrogen) at 4°C overnight on the orbital shaker. The immune-complexes were captured by adding 50 μL of protein A agarose bead slurry (50% v/v) and gently rocking on the orbital shaker at 4°C for 2 hours. After pulse centrifugation at 14,000 rpm for 8 seconds, the immunecomplex-protein-A-agarose-bead pellets were collected and washed 5 times with 800 μL of ice-cold modified radioimmunoprecipitation (RIPA) buffer (50 mM Tris-HCl [pH 7.4]; 150 mM NaCl; 1% NP-40; 0.25% sodium deoxycholate; 1 mM EDTA; 1 mM PMSF; 2 μg/mL leupeptin; 2 μg/mL pepstatin A; 2 μg/mL aprotinin; 500 μg/mL benzamidine; 1 mM Na3VO4; 1 mM NaF) and once with 800 μL ice-cold 1× PBS. The pellets containing immune complexes were resuspended in 60 μL of 2× sample buffer (100 mM Tris-HCl [pH 6.8], 200 mM DTT, 4% SDS, 0.2% Bromophenol Blue, 20% Glycerol).
Immunoprecipitates or nuclear proteins were resolved (50 μg of protein per lane) on pre-cast 10% NuPAGE gels (Invitrogen). After electrophoresis, the proteins were transferred to Hybond P nylon membrane (Amersham Biosciences, Piscataway, NJ) at 30 V overnight. The protocol for immunobloting was essentially as detailed elsewhere . Rabbit polyclonal antibody anti-MIXL1-N  was used at a dilution of 1:100 initially or 1:500 for immunoblotting studies. Mouse monoclonal antibody anti-flag M2 (Sigma) 1:300; mouse monoclonal antibody 4G10 (Upstate) 1:3000 or 1:4000.
Alkaline phosphatase treatment
Nuclear extracts of HEK 293T cells transfected with CMV5-MIXL1 was prepared without the addition of phosphatase inhibitors sodium fluoride and sodium orthovanadate. Immediately after extraction, 15 μg of nuclear protein was incubated with 40 units of calf intestine alkaline phosphatase (CIAP, Roche) in a 20 μL reaction for 30 minutes at 30°C. As a control, the same reaction was performed in the presence of 100 mM sodium orthovanadate, which inhibits CIAP activity. In addition, a mock reaction was also performed with no CIAP. The reactions were terminated with NuPAGE sample buffer (Invitrogen). The changes in protein mobility were determined by probing the immunoblots with the anti-MIXL1-N antibody.
Results and discussion
MIXL1 is phosphorylated at multiple sites
MIXL1 is tyrosine phosphorylated
Amino terminal Tyr20 is phosphorylated
Absence of PRD causes a marked reduction in tyrosine phosphorylation
Since MIXL1 localizes to the predominantly to the nucleus (Guo and Nagarajan unpublished results), the kinase(s) responsible for tyrosine phosphorylation on MIXL1 is likely to be localized in the nucleus. Ten out of the 90 tyrosine kinases encoded in humans, are known to localize to the nucleus (reviewed by Cans et al ). Thus the likely candidates are c-ABL1, Wee1, FRK, LYN, FES family (FES and FER) and JAK family (JAK1, JAK2, JAK3, and TYK2). Although the present studies were conducted in HEK293 cells, several of these kinases are expressed in the hematopoietic system. Additionally, since the tyrosine phosphorylation of MIXL1 was not examined in cytoplasm, we could not rule out that MIXL1 is tyrosine phosphorylated in the cytoplasm and translocated into the nucleus.
Unlike serine/threonine phosphorylation, tyrosine phosphorylation on homeodomain proteins is rarely reported to date. Hence the role of tyrosine phosphorylation in the regulation of homeodomain proteins largely remains unclear. The only reported case is the tyrosine phosphorylation of HoxA10 during interferon γ-induced myeloid differentiation. In this case, interferon γ-induced differentiation led to HoxA10 tyrosine phosphorylation in the myelomonocytic cell line U937, which decreased DNA binding of HoxA10 to Pbx-HoxA10 binding sites . However, SHP1 protein-tyrosine phosphatase (SHP1-PTP), which antagonizes myeloid differentiation, decreased tyrosine phosphorylation of HOXA10 homeodomain thereby enhancing HOXA10-mediated repression .
A tissue- or cell cycle-specific phosphorylation may alter MIXL1 activity. Future studies will elucidate whether phosphorylation mediated protein-protein interactions due to the unique PRD in human, mouse and chicken Mix-like proteins indeed substitutes for the functional diversity achieved by multiple members in Xenopus Laevis.
We thank support from Abraham J and Phyllis Katz foundation. The University of Texas M.D. Anderson Cancer Center DNA Sequencing Facility is supported by NIH core grant CA-16672.
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