Critical Role for TCR Signal Strength and MHC Specificity in ThPOK-Induced CD4 Helper Lineage Choice.

Sustained TCR signaling is critical for ThPOK induction in MHC class II (MHCII)-signaled thymocytes leading to the CD4 helper lineage commitment. ThPOK suppresses the cytotoxic program in the signaled thymocytes and is shown to be necessary and sufficient for the CD4 helper lineage choice. Accordingly, loss and gain of ThPOK function redirects MHCII- and MHC class I (MHCI)-signaled thymocytes into the CD8 cytotoxic and CD4 helper lineage, respectively. However, the impact of a defined ThPOK level on the CD4 helper lineage choice of MHCII- and MHCI-specific thymocytes and the role of TCR signaling in this process is not evaluated. Equally, it is not clear if suppression of the cytotoxic program by ThPOK is sufficient in redirecting MHCI-restricted thymocytes into the CD4 helper lineage. In this study, we have investigated CD8 to CD4 helper lineage redirection in three independent ThPOK overexpressing transgenic mouse lines. Our analysis shows that one of the transgenic lines, despite overexpressing ThPOK compared with wild-type CD4 mature T cells and compromising cytotoxic program, failed to redirect all MHCI-signaled thymocytes into the CD4 helper lineage, resulting in the continued presence of CD8+ mature T cells and the generation of a large number of double negative mature T cells. Critically, the same ThPOK transgene completely restored the CD4 helper lineage commitment of MHCII-specific Thpok -/- thymocytes. Importantly, augmenting TCR signaling significantly enhanced the ThPOK-mediated CD4 helper lineage choice of MHCI-specific thymocytes but was still substantially less efficient than that of MHCII-specific thymocytes expressing the same amount of ThPOK. Together, these data suggest that the ThPOK-induced CD4 helper lineage commitment is strongly influenced by TCR signal strength and MHC specificity of developing thymocytes.

MAP KINASE PHOSPHATASE1 Controls Cell Fate Transition during Stomatal Development.

Stomata on the plant epidermis control gas and water exchange and are formed by MAPK-dependent processes. Although the contribution of MAP KINASE3 (MPK3) and MPK6 (MPK3/MPK6) to the control of stomatal patterning and differentiation in Arabidopsis (Arabidopsis thaliana) has been examined extensively, how they are inactivated and regulate distinct stages of stomatal development is unknown. Here, we identify a dual-specificity phosphatase, MAP KINASE PHOSPHATASE1 (MKP1), which promotes stomatal cell fate transition by controlling MAPK activation at the early stage of stomatal development. Loss of function of MKP1 creates clusters of small cells that fail to differentiate into stomata, resulting in the formation of patches of pavement cells. We show that MKP1 acts downstream of YODA (a MAPK kinase kinase) but upstream of MPK3/MPK6 in the stomatal signaling pathway and that MKP1 deficiency causes stomatal signal-induced MAPK hyperactivation in vivo. By expressing MKP1 in the three discrete cell types of stomatal lineage, we further identified that MKP1-mediated deactivation of MAPKs in early stomatal precursor cells directs cell fate transition leading to stomatal differentiation. Together, our data reveal the important role of MKP1 in controlling MAPK signaling specificity and cell fate decision during stomatal development.

Negative Regulation of Zap70 by Lck Forms the Mechanistic Basis of Differential Expression in CD4 and CD8 T Cells.

Lck and Zap70, two non-receptor tyrosine kinases, play a crucial role in the regulation of membrane proximal TCR signaling critical for thymic selection, CD4/CD8 lineage choice and mature T cell function. Signal initiation upon TCR/CD3 and peptide/MHC interaction induces Lck-mediated phosphorylation of CD3 ITAMs. This is necessary for Zap70 recruitment and its phosphorylation by Lck leading to full Zap70 activation. In its native state Zap70 maintains a closed conformation creating an auto-inhibitory loop, which is relieved by Lck-mediated phosphorylation of Y315/Y319. Zap70 is differentially expressed in thymic subsets and mature T cells with CD8 T cells expressing the highest amount compared to CD4 T cells. However, the mechanistic basis of differential Zap70 expression in thymic subsets and mature T cells is not well understood. Here, we show that Zap70 is degraded relatively faster in DP and mature CD4 T cells compared to CD8 T cells, and inversely correlated with relative level of activated Zap70. Importantly, we found that Zap70 expression is negatively regulated by Lck activity: augmented Lck activity resulting in severe diminution in total Zap70. Moreover, Lck-mediated phosphorylation of Y315/Y319 was essential for Zap70 degradation. Together, these data shed light on the underlying mechanism of Lck-mediated differential modulation of Zap70 expression in thymic subsets and mature T cells.

MKP1 acts as a key modulator of stomatal development.

The MAPK signaling cascade is universal among eukaryotes and mediates a variety of environmental and developmental responses. Two Arabidopsis MAPKs, MPK3 and MPK6, have been shown to be activated by various stimuli and suggested as a convergence point of different signaling pathways. It is known that these MAPKs, MPK3/MPK6, control the discrete stages of stomatal development in Arabidopsis, but how they are regulated and how the same MAPK components can achieve signaling specificity is largely unknown. We recently demonstrated that MAP Kinase Phosphatase 1 (MKP1) promotes stomatal differentiation by suppressing activation of MPK3/MPK6 in the stomatal lineage. By expressing MKP1 in discrete stomatal precursor cell types, we further identified that MKP1 plays an important role at the early stage of stomatal development for the cell fate transition leading to stomatal differentiation. While MKP1 was previously known as a key regulator of environmental stress responses, our data illustrate a novel role of MKP1 in plant development: it acts as one of the specificity-determining regulators of MAPK signaling to enforce proper stomatal development in Arabidopsis.