AKT and CDC37 activation is unaffected by loss of CD5-CK2 signaling pathway.

<p>Scatter plot of pAKT(S473) levels (A) and pCDC37 (S13) levels (B) within DP or CD4+SP thymocytes obtained from CD5-WT OTII and CD5-ΔCK2BD OTII TCR-Tg mice after injection with PBS (control) or 300 μg of OVAp. Data represents 4 independent experiments (n = 5 mice each group). NS = not significant, unpaired two-tailed Student-t test.</p

Jak3 is required for actin polymerization in murine T lymphocytes stimulated with CCL21.

<p>Cells were stimulated with 300/mL of the chemokine CCL21 for 0–300 seconds and F-actin was detected by staining with NBD-phallacidin-FITC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088014#s2" target="_blank">materials and methods</a>. Representative histograms (<i>top</i>) and graph (<i>bottom</i>) of F-actin increment are shown. <i>A</i>, Wild type (blue line) or Jak3-deficient (red line) lymphocytes were stimulated with the chemokine. Graph represents the average of 4 independent experiments ± SEM. Statistical significance was calculated using an unpaired Student's t-test (one-tailed). Asterisks indicate *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. <i>B</i>, Primary lymphocytes from C57BL/6 mice pre-treated with DMSO (blue line), WHI-P131 (red line) or PTX (green line), stimulated with the chemokine at the same time points. Representative histograms (<i>top</i>) and graph (<i>bottom</i>) of the F-actin increment are shown. The graph represents the average values of 9 independent experiments ± SEM. *<i>p</i><0.05, **<i>p</i><0.01. Statistical significance was determined with a paired Student's t-test (one-tailed).</p

Jak3 inhibition affects cofilin phosphorylation in response to chemokines.

<p><i>A</i>, PLN lymphocytes from C57BL/6 mice pre-treated with DMSO, WHI-P131 or PTX, were stimulated for 0 to 300 seconds with CCL21 and p-cofilin levels were analyzed at indicated time points. Cells were lysed, supernatants were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088014#s2" target="_blank">materials and methods</a> and western blots were performed with anti phospho-cofilin antibody. Anti-actin antibody was used as loading control. Pooled PLN lymphocytes from 4 mice were used in each assay. One representative experiment (of a total of 3) is shown. <i>B</i>, Primary human PBMCs with the same pre-treatments were stimulated with CXCL12 for 0 to 300 seconds and p-cofilin analysis was performed at the indicated time points. Densitometric analysis of the blots was performed as described above for each group. One healthy donor was used for each experiment. A representative experiment is shown (n = 3).</p

Nuclear translocation pERK is independent of CD5-CK2 signaling.

<p>(A) Images of DP and CD4+SP thymocytes obtained from PBS or OVAp injected CD5-WT OTII and CD5-ΔCK2BD OTII TCR-Tg mice and analyzed by imaging flow cytometry. The images show each channel individually represented as well as an overlay of all channels. The data are representative of at least 900 images each from 2 independent experiments (n = 2 mice each group). (B) Quantitative representation of all events represented in (A). The graphs reflect mean±SEM pERK levels inside the nucleus (left panels) or ratio of pERK in nucleus to whole cell (right panels) within DP (upper panels) or CD4+SP (lower). (C) Images of pERK in DP and CD4+SP thymocytes obtained from non-TCR transgenic CD5-WT and CD5-ΔCK2BD mice. The images are representative of 900–1500 images from 3 independent experiments (n = 3 mice each group). (D) Quantitative representation analyzed similar to (B). **p<0.01 ****p<0.0001, unpaired two-tailed Student-t test.</p

CD5-CK2 Signaling Modulates Erk Activation and Thymocyte Survival

<div><p>CD5 is well recognized for its importance in thymic selection. Although this property of CD5 has been attributed to its ITIM-domain dependent regulation of TCR-signal strength, the mechanism has not been established. A second major signaling domain within the cytoplasmic tail of CD5 is a CK2 binding/activation domain (CD5-CK2BD). Using a gene-targeted mouse in which the CD5-CK2BD is selectively ablated (CD5-ΔCK2BD), we determined that loss of function of CD5-CK2 signaling in a MHC-II selecting TCR transgenic (OT-II) mouse resulted in decrease in double positive (DP) thymocytes, which correlated with enhanced apoptosis. Remarkably, DP cells expressing high levels of CD5 and CD69 and single positive (CD4+SP) thymocytes were increased in CD5-ΔCK2BD mice indicating that CD5-CK2 signaling regulates positive selection and promotes survival. Consistent with this possibility, we determined that the activation and nuclear localization of ERK as well as apoptosis was greater in thymic populations from OTII CD5-ΔCK2BD mice than OTII CD5-WT mice following injection of OVA_323-339-peptide. The mobilization of Ca²⁺, an early event of TCR activation, was not altered by the loss of CD5-CK2 signaling. Collectively, these data demonstrate that the CD5-CK2 signaling axis regulates positive selection by modulating activation of ERK and promoting survival independent of proximal TCR signals.</p></div

Jak3 Enables Chemokine-Dependent Actin Cytoskeleton Reorganization by Regulating Cofilin and Rac/Rhoa GTPases Activation

<div><p>We have previously shown that Jak3 is involved in the signaling pathways of CCR7, CCR9 and CXCR4 in murine T lymphocytes and that Jak3^−/− lymphocytes display an intrinsic defect in homing to peripheral lymph nodes. However, the molecular mechanism underlying the defective migration observed in Jak3^−/− lymphocytes remains elusive. Here, it is demonstrated for the first time, that Jak3 is required for the actin cytoskeleton reorganization in T lymphocytes responding to chemokines. It was found that Jak3 regulates actin polymerization by controlling cofilin inactivation in response to CCL21 and CXCL12. Interestingly, cofilin inactivation was not precluded in PTX- treated cells despite their impaired actin polymerization. Additionally, Jak3 was required for small GTPases Rac1 and RhoA activation, which are indispensable for acquisition of the migratory cell phenotype and the generation of a functional leading edge and uropod, respectively. This defect correlates with data obtained by time-lapse video-microscopy showing an incompetent uropod formation and impaired motility in Jak3-pharmacologically inhibited T lymphocytes. Our data support a new model in which Jak3 and heterotrimeric G proteins can use independent, but complementary, signaling pathways to regulate actin cytoskeleton dynamics during cell migration in response to chemokines.</p></div

Ablation of CD5-CK2 binding domain alters thymic T cell development.

<p>Thymic DN, DP (CD4+CD8+) and SP (CD4+ or CD8+) populations (dot plots), proportions and absolute numbers (graphs) in CD5-WT OTII and CD5-ΔCK2BD OTII TCR-Tg mice (A) and CD5-WT and CD5-ΔCK2BD mice (B). Ratio of CD4:CD8 SP cells in CD5-WT OTII and CD5-ΔCK2BD OTII TCR-Tg mice (C) and CD5-WT and CD5-ΔCK2BD non-TCR Tg mice (D). The flow cytometric dot plots are representative of one of five mice from each strain. The graphs represent data from 4 independent experiments (n = 4–7 each group) *p<0.05, **p<0.01, ***p<0.001, unpaired two-tailed Student-t test.</p

Increased TCR dependent MAPK activation and enhanced AICD in CD5-CK2BD deficient mice is independent of peripheral T cells activation and thymocyte FasL expression.

<p>(A) Total cell numbers (left panel) of CD5-WT vs CD5-ΔCK2BD newborn mice after injection of either PBS (control) or α-CD3 (20 μg) for 24h. To evaluate the effect of anti-CD3 stimulation, both CD5-ΔCK2BD or CD5-WT were compared with their respective unstimulated controls. <b>S</b>ubpopulation analysis show DP (middle panel) or CD4+ SP thymocytes (right panel) (B) Scatter plots showing Bcl-2 levels in DP and CD4+ SP thymocytes. (C) Bar graphs of FasL expression in PBS injected and anti-CD3 injected mice. Adult thymocytes were used as positive control. (D) Scatter plots show pERK expression 24h after α-CD3 injection or PBS as in DP and CD4+ SP cells from CD5-WT and CD5-ΔCK2BD newborn mice. (E) Graphs showing expression of pJNK (F) pP38 and (G) pZap70. Each graph is representative of 1 independent experiment (n = 5–6 each group). *p<0.05, **p<0.01, ***p<0.001, unpaired two-tailed Student-t test.</p

Jak3 and G protein play complementary and independent roles in chemokine receptor mediated signaling.

<p>Chemokine receptor activates both signaling pathways G_αi (green lines) and Jak3 (yellow lines). Dotted lines indicate proposed pathways, while continuous lines indicate already reported pathways. First, G_αi, activates Cdc42 and Arp2/3 complex generating membrane protrusions through actin bundles leading to filopodia formation. Then, Jak3 is activated independently of G_αi and both lead to Rac1 activation and its association with the Arp2/3 complex, driving rearrangement of the actin network to for lamellipodia. This last step is accompanied of SSH1L activation which is required for dephosphorylation of both cofilin (activation) and LIMK1 (inactivation). Cofilin activation elicits free sites for Arp2/3 complex association with the actin filaments allowing branched actin polymerization which contributes to the assembly of the actin network. Next, both Jak3 and G_αi, are required for RhoA activation, which leads to activation of downstream effectors ROCK and MLC, resulting in the actomyosin complex assembly and function. At the same time, cofilin is phosphorylated by LIMK1 at the leading edge, which formation depends on Jak3 but no on G protein activity. Also, LIMK2 activation is a later event dependent on ROCK that in turn inactivates cofilin at the rear end of the cell and prevents actomyosin complex disassociation and uropod formation.</p

Rac1 activation is diminished in Jak3-inhibited or G_αi-inactivated T lymphocytes after CCL21 stimulation.

<p><i>A</i>, Representative images are shown from DMSO-, WHI-P131- or PTX-treated cells stimulated for 0, 30 and 300 seconds with CCL21. One representative cell stained with Rhodamine-phalloidin (F-actin) and Rac1-GTP Alexa-Fluor 488 (activated Rac1) is shown for each condition. <i>B</i>, The graph represents the average of mean fluorescence intensity measurements of single cells (between 5-26 cells per coverslip). An average of 36 cells per condition were individually analyzed for GTPase activation from each experiment. Data are expressed as relative increment (RI) of the fluorescence in each sample compared to unstimulated control cells. Mean values ± SEM from 3 independent experiments are shown. Asterisks indicate statistical significance (*<i>p</i><0.05).</p