RhoH GTPase recruits and activates Zap70 required for T cell receptor signaling and thymocyte development

RhoH-deficient animals show impaired T-cell differentiation due to defective T-cell receptor signalling.

“On the other hand, animals showed impaired T-cell differentiation due to defective T-cell receptor signalling [21,28], as RhoH is necessary for the production and survival or migration of T-cells.”

RhoH activity is modulated by interaction with other Rho GTPases, transcriptional regulation, or phosphorylation rather than through classical GTPase cycling and effector modulation.

“Its activity is modulated by interaction with other Rho GTPases [19,20], transcriptional regulation [19] or phosphorylation [21] rather than through the classical cycling process and modulation by effector proteins.”

RhoH is necessary for the production and survival or migration of T-cells.

“On the other hand, animals showed impaired T-cell differentiation due to defective T-cell receptor signalling [21,28], as RhoH is necessary for the production and survival or migration of T-cells.”

RhoH-deficient mice do not develop lymphomas and have no obvious defects in HSC maintenance, but show impaired T-cell differentiation attributed to defective T-cell receptor signalling.

“RhoH-deficient mice did not develop lymphomas and had no obvious defects in HSC maintenance, but they showed impaired T-cell differentiation attributed to defective T-cell receptor signalling.”

Zap70 is a specifically interacting protein partner of RhoH downstream of the TCR.

“A specifically interacting protein was analysed by mass spectrometry and found to be Zap70.”

Injection of irradiated mice with bone marrow cells transduced with RhoH confirmed the crucial role of RhoH in thymocyte maturation and TCR signalling, since reconstitution of RhoH compensated for the defects of RhoH-deficient mice.

“Injection of irradiated mice with bone marrow cells transduced with RhoH confirmed the crucial role of RhoH in thymocyte maturation and TCR signalling since reconstitution of RhoH compensated for the defects of RhoH-deficient mice.”

RhoH-deficient mice show impaired development of thymocytes and reduced TCR signalling due to inefficient recruitment of Zap70.

“RhoH-deficient mice showed an impaired development of thymocytes and reduced TCR signalling due to inefficient recruitment of Zap70 [21,28].”

Mutational analysis confirmed that the two tyrosines Y73 and Y83 in RhoH are essential for recruitment of Zap70.

“Further mutational analysis confirmed the theory that the two tyrosines Y73 and Y83 in RhoH are essential for recruitment of Zap70.”

Deletion of the two SH2 domains in Zap70 known to bind to the TCR CD3ζ chain abolishes interaction with RhoH.

“Deletion of the two SH2 domains in Zap70 known to bind to the TCR CD3ζ chain also abolished interaction with RhoH.”

Stimulation of Jurkat cells with anti-CD3ε increases tyrosine phosphorylation of RhoH.

“Indeed, the authors found that stimulation of Jurkat cells with anti-CD3ε increased tyrosine phosphorylation of RhoH.”

RhoH acts as a positive regulatory factor for thymocyte selection and T-cell receptor signalling.

“Two groups independently showed that RhoH acts as a positive regulatory factor for thymocyte selection and T-cell receptor signalling [21,28].”

RhoH contains a novel ITAM-like motif that serves as a binding site for Zap70, which is then shuttled to the T-cell receptor complex where it triggers activation of the LAT signalosome.

“RhoH contains a novel ITAM-like motif that serves as a binding site for Zap70, which is then shuttled to the T-cell receptor complex where it serves as a crucial molecule to trigger activation of the LAT signalosome.”

The physical interaction of RhoH and Zap70 was confirmed in coimmunoprecipitation studies overexpressing the two proteins in HEK293 cells.

“The physical interaction of RhoH and Zap70 was confirmed in coimmunoprecipitation studies overexpressing the two proteins in HEK293 cells.”

In RhoH-deficient mice, the number of double negative thymocytes is reduced by 60%, indicating disturbed development between DN and DP stages; this is reflected in an elevated number of CD44- CD25+ DN3 cells.

“In mutant mice, the number of double negative thymocytes was reduced by 60%, indicating that the development between double negative (DN) to double positive (DP) stages was disturbed; this was also reflected in an elevated number of CD44- CD25+ DN3 cells.”

The phenotype of RhoH-deficient mice does not suggest a defect in maintenance of haematopoietic progenitors.

“However, the phenotype of the RhoH-deficient mice does not suggest that the mice have a defect in maintenance of haematopoietic progenitors [21,28].”

RhoH acts as a shuttle for Zap70, interacting with cytoplasmic Zap70 in the absence of antigen presenting cells and moving to the plasma membrane upon activation.

“Subcellular fractionation and immunofluorescence staining showed that RhoH acts as a shuttle for Zap70 and interacts with the pool of Zap70 molecules localised in the cytoplasm in the absence of antigen presenting cells.”

RhoH contains an ITAM-like motif where the (iso)leucines at position +3 are replaced by an alanine.

“A closer look at the RhoH sequence revealed that RhoH contains a sequence that the authors dubbed an ITAM-like motif, where the (iso)leucines at position +3 are replaced by an alanine.”

The Zap70-RhoH complex is detectable in unstimulated, resting cells.

“However, while stimulation was a prerequisite for the interaction of tyrosine phosphorylated Zap70 with the TCR, the Zap70-RhoH complex was also detectable in unstimulated, resting cells.”

RhoH-deficient mice have abnormally low levels of T-lymphocytes and thymus cellularity approximately 60% lower than control animals, due to reduced proliferative cell expansion.

“RhoH-deficient mice were found to have abnormally low levels of T-lymphocytes and the cellularity of their thymus was app. 60% lower than in control animals, due to reduced proliferative cell expansion.”

Although peripheral thymocytes express the TCR complex at normal levels, anti-CD3ε stimulation triggers only a reduced proliferative response, suggesting down-regulation occurs downstream of the TCR complex.

“Although peripheral thymocytes expressed the TCR complex at normal levels, anti-CD3ε stimulation triggered only a reduced proliferative response suggesting that the down-regulation occurred downstream of the TCR complex [21].”

In RhoH-deficient mice, expression of positive selection markers CD5 and CD69 is reduced, suggesting RhoH plays a role in thymocyte positive selection.

“In RhoH-deficient mice the expression of these markers was reduced, suggesting that RhoH plays a role in thymocyte positive selection.”

Phosphorylation of LAT, VAV1, PLCγ and Erk is reduced in RhoH-deficient DP cells.

“Indeed, phosphorylation of LAT, VAV1 and of PLCγ ανδ Erk, were reduced in RhoH-deficient DP cells [21,28].”

The ITAM-like motif is absent in any of the other Rho GTPases.

“Interestingly, this motif is absent in any of the other Rho GTPases.”

RHOH is involved in T-cell receptor signaling through the recruitment and activation of ZAP70 and LCK.

“RHOH is involved in T-cell receptor signaling through the recruitment and activation of ZAP70 and LCK (4,5).”

In RHOH WT-expressing Jurkat cells, ZAP70 could bind with RHOH in a resting state, and RHOH-bound ZAP70 increased after anti-CD3 stimulation.

“In RHOH WTexpressing Jurkat cells, ZAP70 could bind with RHOH in a resting state, and RHOH-bound ZAP70 increased after anti-CD3 stimulation, consistent with previous publications (5).”

The function of RHOH depends on the phosphorylation of its ITAMs.

“The function of RHOH depends on the phosphorylation of its ITAMs (5).”

Studies on RhoH-/- mice have observed a defect in thymocyte selection, maturation, lower T-cell cytotoxicity and cytokine release, and demonstrated that it is a result of reduced activation of ZAP70-mediated signaling and impaired translocation of ZAP70 to the immunological synapse.

“Studies on RhoH -/-mice have observed a defect in thymocyte selection, maturation, lower T-cell cytotoxicity and cytokine release, and have demonstrated that it is a result of reduced activation of ZAP70-mediated signaling and impaired translocation of ZAP70 to the immunological synapse (5,9).”