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. 2009 Jul 15;183(2):1215-21.
doi: 10.4049/jimmunol.0803887. Epub 2009 Jun 24.

The polarity protein Par1b/EMK/MARK2 regulates T cell receptor-induced microtubule-organizing center polarization

Joseph Lin  1 , Kirk K Hou, Helen Piwnica-Worms, Andrey S Shaw
Affiliations

The polarity protein Par1b/EMK/MARK2 regulates T cell receptor-induced microtubule-organizing center polarization

Joseph Lin et al. J Immunol. .

Abstract

Engagement of a T cell to an APC induces the formation of an immunological synapse as well as reorientation of the microtubule-organizing center (MTOC) toward the APC. How signals emanating from the TCR induce MTOC polarization is not known. One group of proteins known to play a critical role in asymmetric cell division and cell polarization is the partitioning defective (Par) family of proteins. In this study we found that Par1b, a member of the Par family of proteins, was inducibly phosphorylated following TCR stimulation. This phosphorylation resulted in 14-3-3 protein binding and caused the relocalization of Par1b from the membrane into the cytoplasm. Because a dominant-negative form of Par1b blocked TCR-induced MTOC polarization, our data suggest that Par1b functions in the establishment of T cell polarity following engagement to an APC.

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Figures

Figure 1
Figure 1. Par1b is inducibly phosphorylated following TCR stimulation at S400 and T595
A) Jurkat T cells were transfected with FLAG-tagged Par1b and then stimulated with an anti-TCR mAb for the indicated time points. Cells were then lysed and protein samples immunoprecipitated with an anti-FLAG antibody. Precipitates were separated by SDS-PAGE and visualized by blotting with phosphospecific antibodies to S400 and T595. Samples were also blotted with anti-FLAG to show equal protein levels. B) Endogenous Par1b was immunoprecipitated from 6×107 Jurkat cells that were either stimulated with anti-TCR mAb or left unstimulated. Immunoprecipitates were divided into 3 equal samples, separated by SDS-PAGE, and visualized by immunoblotting for pS400, pT595, and Par1b. Note that there are two bands for endogenous Par1b due to alternative splicing. C) Cells transfected with FLAG-tagged Par1b were pretreated for 30 min. with the Src kinase inhibitor PP2 prior to stimulation with an anti-TCR antibody. Samples were assayed as in A.
Figure 2
Figure 2. Expression of PKCs induces phosphorylation of Par1b at S400 and T595
A) FLAG-wt Par1b was transfected with myristoylated (myr) or kinase-dead (KD) PKCζ. Samples were then blotted with phosphospecific antibodies and anti-PKCζ to show expression. B) FLAG-wt Par1b was transfected with constitutively active (CA) or kinase-dead (KD) mutants of Xpress-tagged PKCθ, PKCα, or a vector control. Cells were then lysed, Par1b immunoprecipitated, and the phosphorylation state of S400 and T595 determined by blotting with phosphospecific antibodies. Samples were also blotted with anti-FLAG and anti-Xpress to demonstrate protein production.
Figure 3
Figure 3. S400 and T595 mediate 14-3-3 protein binding and Par1b localization
A) Jurkat T cells were transfected with myc-tagged 14-3-3 and various Par1b constructs (wt or mutated at S400 and/or T595). Cells were then lysed and Par1b immunoprecipitated. Samples were then blotted with anti-myc and anti-FLAG antibodies. Cell lysates were blotted to demonstrate equal 14-3-3 protein expression. B) Jurkat T cells were transfected with the indicated Par1b constructs and then stimulated via the TCR or left unstimulated for 5 min. Cells were then lysed and Par1b immunoprecipitated. Samples were then analyzed by blotting for endogenous 14-3-3 associated with Par1b. C) Cells were transfected with either wt Par1b or the S400/T595 double mutant (ST/AA). Cells were then fractionated into membrane or cytoplasmic fractions and immunoblotted with the indicated antibodies.
Figure 4
Figure 4. Par1b localizes to the cytoplasm and membrane at T cell:APC contact site
A) Jurkat T cells were transfected with GFP-wt Par1b or GFP-KD Par1b and conjugated to Daudi B cells incubated with SEE. Cells were then fixed in 3% PFA and visualized by confocal microscopy. B) Conjugates were also stained with anti-CD3 (red) to examine cytoplasmic vs. membrane localization of Par1b (green) at the contact site. C) Par1b accumulation in the cytoplasm was quantitated in more than 100 conjugates per experiment. Shown are the average and standard deviation of two experiments. D) GFP-wt Par1b transfected Jurkat T cells were pre-incubated with 10 µM PP2 prior to conjugate formation and visualized as before. E) OT-1 T cells were transduced with a retrovirus encoding FLAG-tagged Par1b. Conjugates with peptide-loaded (1 µM SIINFEKL) RMA-S cells were then formed and Par1b localization determined by immunofluorescence with anti-FLAG. Bar 5 µm.
Figure 5
Figure 5. Dominant Negative Par1b blocks TCR induced MTOC polarization
A) Jurkat T cells transfected with FLAG-tagged KD-Par1b were stimulated with Daudi cells in the presence of 1 µg/ml SEE for 20 minutes. Cells were then fixed with 3%PFA and stained with the anti-FLAG (red) to indicate transfected cells, anti-tubulin (green) to visualize the position of the MTOC and anti-human Ig (blue) to denote the APC. Two conjugates are shown: the upper conjugate is an untransfected T cell showing normal MTOC polarization. The T cell in the lower conjugate (denoted by arrowhead), expressing KD-Par1b, shows defective MTOC polarization. B) KD-Par1b transfected Jurkat T cells were stimulated on anti-TCR coated coverslips and allowed to polarize for 20 minutes before fixation in 3% PFA. Cells were then stained with anti-FLAG to identify transfected cells and anti-tubulin to visualize the position of the MTOC. Arrowheads denote cells expressing KD-Par1b. Confocal sections adjacent to the coverslip were visualized and quantitated. No anti-TCR on the coverslip (No Stim) and a control transfection of an unrelated FLAG-tagged protein (Control) were negative and positive controls for polarization respectively. Five experiments in which over 100 cells were scored in a blinded manner were averaged. Error bars represent standard deviation. Bar 5 µm. C) Primary T cells were transfected with KD-Par1b-GFP (green) and stimulated on anti-CD3 coated coverslips for 20 min. Cells were then fixed and stained with anti-tubulin (red) to visualize the position of the MTOC. The average of three experiments in which over 30 cells were scored is shown. Error bars represent standard deviation.
Figure 6
Figure 6. Model of Par1b
A) In a resting T cell, Par1b is associated with the plasma membrane whereas 14-3-3 proteins, PKCs and PKD reside in the cytoplasm. At the membrane, Par1b can phosphorylate substrates such as Par3 preventing association with the membrane. B) Following engagement to an APC, PKCζ, PKCθ, and PKD are recruited to the membrane at the contact site termed the immunological synapse. There, these kinases phosphorylate Par1b at S400 and T595 allowing 14-3-3 proteins to bind, causing relocalization into the cytoplasm where Par1b plays a role in MTOC polarization. Once Par1b is disassociated from the membrane, Par3 can accumulate at the contact site. Grey arrows depict movement, black arrows indicate phosphorylation events, and empty arrowheads indicate binding.
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