CBAP regulates the function of Akt-associated TSC protein complexes to modulate mTORC1 signaling.

The Akt-Rheb-mTORC1 pathway plays a crucial role in regulating cell growth, but the mechanisms underlying the activation of Rheb-mTORC1 by Akt remain unclear. In our previous study, we found that CBAP was highly expressed in human T-ALL cells and primary tumors, and its deficiency led to reduced phosphorylation of TSC2/S6K1 signaling proteins as well as impaired cell proliferation and leukemogenicity. We also demonstrated that CBAP was required for Akt-mediated TSC2 phosphorylation in vitro. In response to insulin, CBAP was also necessary for the phosphorylation of TSC2/S6K1 and the dissociation of TSC2 from the lysosomal membrane. Here we report that CBAP interacts with AKT and TSC2, and knockout of CBAP or serum starvation leads to an increase in TSC1 in the Akt/TSC2 immunoprecipitation complexes. Lysosomal-anchored CBAP was found to override serum starvation and promote S6K1 and 4EBP1 phosphorylation and c-Myc expression in a TSC2-dependent manner. Additionally, recombinant CBAP inhibited the GAP activity of TSC2 complexes in vitro, leading to increased Rheb-GTP loading, likely due to the competition between TSC1 and CBAP for binding to the HBD domain of TSC2. Overexpression of the N26 region of CBAP, which is crucial for binding to TSC2, resulted in a decrease in mTORC1 signaling and an increase in TSC1 association with the TSC2/AKT complex, ultimately leading to increased GAP activity toward Rheb and impaired cell proliferation. Thus, we propose that CBAP can modulate the stability of TSC1-TSC2 as well as promote the translocation of TSC1/TSC2 complexes away from lysosomes to regulate Rheb-mTORC1 signaling.

A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma.

Chimeric antigen receptor T cell (CAR-T) therapy has been applied in the treatment of B-cell lymphoma; however, CAR-T manufacturing requires virus- or non-virus-based genetic modification, which causes high manufacturing costs and potential safety concerns. Antibody-cell conjugation (ACC) technology, which originated from bio-orthogonal click chemistry, provides an efficient approach for arming immune cells with cancer-targeting antibodies without genetic modification. Here, we applied ACC technology in Vγ9Vδ2 T (γδ2 T) cells to generate a novel off-the-shelf CD20-targeting cell therapy ACE1831 (rituximab-conjugated γδ2 T cells) against relapsed/refractory B-cell lymphoma. ACE1831 exhibited superior cytotoxicity against B-cell lymphoma cells and rituximab-resistant cells compared to γδ2 T cells without rituximab conjugation. The in vivo xenograft study demonstrated that ACE1831 treatment strongly suppressed the aggressive proliferation of B-cell lymphoma and prolonged the survival of tumor-bearing mice with no observed toxicity. Mass spectrometry analysis indicated that cell activation receptors including the TCR complex, integrins and cytokine receptors were conjugated with rituximab. Intriguingly, the antigen recognition of the ACC-linked antibody/receptor complex stimulated NFAT activation and contributed to ACE1831-mediated cytotoxicity against CD20-expressing cancer cells. This study elucidates the role of the ACC-linked antibody/receptor complex in cytotoxicity and supports the potential of ACE1831 as an off-the-shelf γδ2 cell therapy against relapsed/refractory B-cell lymphoma.

Palmitoyl Acyltransferase Activity of ZDHHC13 Regulates Skin Barrier Development Partly by Controlling PADi3 and TGM1 Protein Stability.

Deficiency of the palmitoyl-acyl transferase ZDHHC13 compromises skin barrier permeability and renders mice susceptible to environmental bacterial infection and inflammatory dermatitis. It had been unclear how the lack of ZDHHC13 proteins resulted in cutaneous abnormalities. In this study, we first demonstrate that enzymatic palmitoylation activity, rather than protein scaffolding, by ZDHHC13 is essential for skin barrier integrity, showing that knock-in mice bearing an enzymatically dead DQ-to-AA ZDHHC13 mutation lost their hair after weaning cyclically, recapitulating knockout phenotypes of skin inflammation and dermatitis. To establish the ZDHHC13 substrates responsible for skin barrier development, we employed quantitative proteomic approaches to identify protein molecules whose palmitoylation is tightly controlled by ZDHHC13. We identified over 300 candidate proteins that could be classified into four biological categories: immunological disease, skin development and function, dermatological disease, and lipid metabolism. Palmitoylation of three of these candidates-loricrin, peptidyl arginine deiminase type III, and keratin fiber crosslinker transglutaminase 1-by ZDHHC13 was confirmed by biochemical assay. Palmitoylation was critical for in vivo protein stability of the latter two candidates. Our findings reveal the importance of protein palmitoylation in skin barrier development, partly by promoting envelope protein crosslinking and the filaggrin processing pathway.

Identification of UAP1L1 as a critical factor for protein O-GlcNAcylation and cell proliferation in human hepatoma cells.

Aged hepatocyte-specific-Mcl-1 knockout (MKO-hep) mice are prone to develop liver tumors mimicking human hepatocellular carcinoma (HCC). Here we reported that a protein named UDP-N-acetylglucosamine pyrophosphorylase-1-like-1 (Uap1l1) is upregulated in the liver of young MKO-hep mice without any macroscopically detectable tumor nodules and is prominently expressed in the hepatic tumors developed in the aged MKO-hep mice. Intriguingly, human UAP1L1 is also significantly upregulated in a distinct subset of HCC tissues and patients with upregulated expression of UAP1L1 appeared to have poor prognosis. Overexpression of UAP1L1 significantly promoted, whereas UAP1L1 knockdown markedly reduced the proliferation of human hepatoma cells both in vitro and in vivo. UAP1L1 shows ~59% sequence identity to UDP-N-acetylglucosamine pyrophosphorylase-1 (UAP1), which is directly involved in the synthesis of the sugar donor (UDP-GlcNac) for N-acetylglucosamine modification (O-GlcNAcylation) of proteins. However, unlike UAP1, UAP1L1 harbors very limited UDP-GlcNAc synthesis activity. Moreover, although both UAP1 and UAP1L1 are required for O-GlcNAc transferase (OGT)-mediated protein O-GlcNAcylation, they appear to function distinctly from each other. UAP1L1 directly interacts with OGT, but does not seem to be an OGT substrate. In addition, UAP1L1 alone is not sufficient to activate OGT activity in vitro, suggesting that UAP1L1 may function together with other proteins to modulate OGT activity in vivo. Lastly, UAP1L1 knockdown attenuated c-MYC O-GlcNAcylation and protein stability, and overexpression of c-MYC significantly rescued the proliferation defect of UAP1L1 knockdown HepG2 cells, suggesting that c-MYC is one downstream target of UAP1L1 that contributes to UAP1L1-mediated cell proliferation, at least in HepG2 cells.

CBAP modulates Akt-dependent TSC2 phosphorylation to promote Rheb-mTORC1 signaling and growth of T-cell acute lymphoblastic leukemia.

High-frequency relapse remains a clinical hurdle for complete remission of T-cell acute lymphoblastic leukemia (T-ALL) patients, with heterogeneous dysregulated signaling profiles-including of Raf-MEK-ERK and Akt-mTORC1-S6K signaling pathways-recently being implicated in disease outcomes. Here we report that GM-CSF/IL-3/IL-5 receptor common ß-chain-associated protein (CBAP) is highly expressed in human T-ALL cell lines and many primary tumor tissues and is required to bolster leukemia cell proliferation in tissue culture and for in vivo leukemogenesis in a xenograft mouse model. Downregulation of CBAP markedly restrains expansion of leukemia cells and alleviates disease aggravation of leukemic mice. Transcriptomic profiling and molecular biological analyses suggest that CBAP acts upstream of Ras and Rac1, and functions as a modulator of both Raf-MEK-ERK and Akt-mTORC1 signaling pathways to control leukemia cell growth. Specifically, CBAP facilitated Akt-dependent TSC2 phosphorylation in cell-based assays and in vitro analysis, decreased lysosomal localization of TSC2, and elevated Rheb-GTP loading and subsequent activation of mTORC1 signaling. Taken together, our findings reveal a novel oncogenic contribution of CBAP in T-ALL leukemic cells, in addition to its original pro-apoptotic function in cytokine-dependent cell lines and primary hematopoietic cells, by demonstrating its functional role in the regulation of Akt-TSC2-mTORC1 signaling for leukemia cell proliferation. Thus, CBAP represents a novel therapeutic target for many types of cancers and metabolic diseases linked to PI3K-Akt-mTORC1 signaling.

CBAP functions as a novel component in chemokine-induced ZAP70-mediated T-cell adhesion and migration.

Activated chemokine receptor initiates inside-out signaling to transiently trigger activation of integrins, a process involving multiple components that have not been fully characterized. Here we report that GM-CSF/IL-3/IL-5 receptor common beta-chain-associated protein (CBAP) is required to optimize this inside-out signaling and activation of integrins. First, knockdown of CBAP expression in human Jurkat T cells caused attenuated CXC chemokine ligand-12 (CXCL12)-induced cell migration and integrin α4ß1- and αLß2-mediated cell adhesion in vitro, which could be rescued sufficiently upon expression of murine CBAP proteins. Freshly isolated CBAP-deficient primary T cells also exhibited diminution of chemotaxis toward CC chemokine ligand-21 (CCL21) and CXCL12, and these chemokines-induced T-cell adhesions in vitro. Adoptive transfer of isolated naive T cells demonstrated that CBAP deficiency significantly reduced lymph node homing ability in vivo. Finally, migration of T cell-receptor-activated T cells induced by inflammatory chemokines was also attenuated in CBAP-deficient cells. Further analyses revealed that CBAP constitutively associated with both integrin ß1 and ZAP70 and that CBAP is required for chemokine-induced initial binding of the talin-Vav1 complex to integrin ß1 and to facilitate subsequent ZAP70-mediated dissociation of the talin-Vav1 complex and Vav1 phosphorylation. Within such an integrin signaling complex, CBAP likely functions as an adaptor and ultimately leads to activation of both integrin α4ß1 and Rac1. Taken together, our data suggest that CBAP indeed can function as a novel signaling component within the ZAP70/Vav1/talin complex and plays an important role in regulating chemokine-promoted T-cell trafficking.

An IL-7 splicing-defect lymphopenia mouse model revealed by genome-wide mutagenesis.

Homeostasis of the hematopoietic system is tightly regulated by an array of cytokines that control proliferation, differentiation and apoptosis of various cell lineages. To identify genes that are essential for hematopoietic homeostasis, we screened C57BL/6 mice that had been genome-wide mutagenized by N-ethyl-N-nitrosourea (ENU) to produce altered blood cell composition. We identified a mutant mouse line with a drastic reduction in the number of T and B cell lineages in lymphatic tissues and peripheral blood, as well as severe atrophy of the thymus and lymph nodes. Genotyping with a genome-wide single nucleotide polymorphism (SNP) marker set mapped the mutant phenotype to chromosome 3A and subsequent direct DNA sequencing revealed a G-to-A point mutation in the splicing donor site of the third exon of the candidate gene for IL-7, a lymphocyte survival cytokine. Such mutation resulted in skipping of exon 3 and production of an internally truncated IL-7 (DeltaE3-IL7). Furthermore, using recombinant proteins produced in a baculoviral system, we demonstrated that DeltaE3-IL7 had no detectable anti-apoptotic activity even at a dose that was 30 times more than that required for a wild-type protein to manifest a full activity in a naïve T cell survival assay. Our data suggest that this mutant mouse line provides an alternative animal model for the study of severe combined immunodeficiency (SCID) syndrome in humans.

MAPK-mediated phosphorylation of GATA-1 promotes Bcl-XL expression and cell survival.

In the interleukin 3-dependent hematopoietic cell line Ba/F3, inhibition of mitogen-activated protein kinase, a member of the MAPK/c-Jun N-terminal kinase/stress-activated protein kinase kinase family that plays an important role in cell growth and death control, rapidly leads to severe apoptosis. However, most of the antiapoptotic substrates of MAPK remain to be identified. Here we report that, upon interleukin-3 stimulation of Ba/F3 cells, the transcription factor GATA-1 is strongly phosphorylated at residue serine 26 by a MAPK-dependent pathway. Phosphorylation of GATA-1 increases GATA-1-mediated transcription of the E4bp4 survival gene without significantly changing the DNA-binding affinity of GATA-1. Further characterization of GATA-1 phosphorylation site mutants revealed that the antiapoptotic function of GATA-1 is strongly dependent upon its phosphorylation at the Ser-26 position and is probably mediated through its up-regulation of Bcl-X(L) expression. Taken together, our data demonstrate that MAPK-dependent GATA-1 phosphorylation is important for its transactivation of the E4bp4 gene, Bcl-X(L) expression and cell survival. Therefore, GATA-1 may represent a novel MAPK substrate that plays an essential role in a cytokine-mediated antiapoptotic response.

Tuning up or down the UV-induced apoptosis in Chinese hamster ovary cells with cell cycle inhibitors.

Exposure to UVC induces apoptosis in Chinese hamster ovary (CHO.K1) cells. While studying the underlying mechanism, we found that a variety of cell cycle inhibitors, including colcemid, hydroxyurea and mimosine, enhance the UV-induced apoptosis in these cells. Such enhancement was not dependent on the cell cycle progression nor was it related to the difference in UV sensitivity at different phases of the cell cycle. The expression of p21(waf1/cip1), a general cyclin-dependent kinase (CDK) inhibitor, was deficient in CHO.K1 cells. Ectopic overexpression of the human p21 markedly increased the survival rates of the UV-irradiated cells in the presence of colcemid. In addition, roscovitine, a small-molecule inhibitor of CDK, also inhibited the UV-induced apoptosis. These observations suggest that deregulation of CDK activity may be critical in the UV-induced apoptosis in CHO.K1 cells.

GATA factors are essential for transcription of the survival gene E4bp4 and the viability response of interleukin-3 in Ba/F3 hematopoietic cells.

E4bp4, a member of the basic region/leucine zipper transcription factor superfamily, is up-regulated by the interleukin-3 (IL-3) signaling pathway and plays an important role in the anti-apoptotic response of IL-3. In this study, we demonstrated that E4bp4 is regulated by IL-3 mainly at the transcriptional level. Promoter analysis revealed that a GATA motif downstream of a major transcription initiation site is essential for E4bp4 expression in the IL-3-dependent Ba/F3 cell line. Gel shift assays demonstrated that both GATA-1 and GATA-2 proteins bind to the E4bp4 GATA site in vitro, and the chromatin immunoprecipitation assay further confirmed the in vivo binding of GATA-1 to the E4bp4 promoter. Overexpression of GATA-1 alone transactivates the E4bp4 reporter, whereas transactivation of the E4bp4 reporter by GATA-2 is dependent on the stimulation of IL-3. Last, we demonstrated that alteration of GATA-1 binding to the GATA site by stably overexpressing GATA-1 or a GATA-1 mutant containing only the DNA-binding domain not only modulates the expression of the E4bp4 gene but also influences apoptosis induced by IL-3 removal. Taken together, our results suggest that the GATA factors play an important role in transducing the survival signal of IL-3, and one of their cellular targets is E4bp4.