Expression of Active Protein Kinase B in T Cells Perturbs Both T and B Cell Homeostasis and Promotes Inflammation

Abstract Development of hematopoietic cells depends on a dynamic actin cytoskeleton. Here we demonstrate that expression of the cytoskeletal regulator WASP, mutated in the Wiskott-Aldrich syndrome, provides selective advantage for the development of naturally occurring regulatory T cells, natural killer T cells, CD4+ and CD8+ T lymphocytes, marginal zone (MZ) B cells, MZ macrophages, and platelets. To define the relative contribution of MZ B cells and MZ macrophages for MZ development, we generated wild-type and WASP-deficient bone marrow chimeric mice, with full restoration of the MZ. However, even in the presence of MZ macrophages, only 10% of MZ B cells were of WASP-deficient origin. We show that WASP-deficient MZ B cells hyperproliferate in vivo and fail to respond to sphingosine-1-phosphate, a crucial chemoattractant for MZ B-cell positioning. Abnormalities of the MZ compartment in WASP−/− mice lead to aberrant uptake of Staphylococcus aureus and to a reduced immune response to TNP-Ficoll. Moreover, WASP-deficient mice have increased levels of “natural” IgM antibodies. Our findings reveal that WASP regulates both development and function of hematopoietic cells. We demonstrate that WASP deficiency leads to an aberrant MZ that may affect responses to blood-borne pathogens and peripheral B-cell tolerance.

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TcR and TcR-CD28 Engagement of Protein Kinase B (PKB/AKT) and Glycogen Synthase Kinase-3 (GSK-3) Operates Independently of Guanine Nucleotide Exchange Factor VAV-1

Journal of Biological Chemistry ◽

10.1074/jbc.m604878200 ◽

2006 ◽

Vol 281 (43) ◽

pp. 32385-32394 ◽

Cited By ~ 28

Author(s):

Joanne E. Wood ◽

Helga Schneider ◽

Christopher E. Rudd

Keyword(s):

T Cells ◽

Protein Kinase ◽

Glycogen Synthase ◽

Protein Kinase B ◽

Guanine Nucleotide Exchange Factor ◽

Glycogen Synthase Kinase 3 ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor

TcRζ/CD3 and TcRζ/CD3-CD28 signaling requires the guanine nucleotide exchange factor (GEF) Vav-1 as well as the activation of phosphatidylinositol 3-kinase, protein kinase B (PKB/AKT), and its inactivation of glycogen synthase kinase-3 (GSK-3). Whether these two pathways are connected or operate independently of each other in T-cells has been unclear. Here, we report that anti-CD3 and anti-CD3/CD28 can induce PKB and GSK-3α phosphorylation in the Vav-1–/– Jurkat cell line J. Vav.1 and in primary CD4-positive Vav-1–/– T-cells. Reduced GSK-3α phosphorylation was observed in Vav-1,2,3–/– T-cells together with a complete loss of FOXO1 phosphorylation. Furthermore, PKB and GSK-3 phosphorylation was unperturbed in the presence of GEF-inactive Vav-1 that inhibited interleukin-2 gene activation and a form of Src homology 2 domain-containing lymphocytic protein of 76-kDa (SLP-76) that is defective in binding to Vav-1. The pathway also was intact under conditions of c-Jun N-terminal kinase (JNK) inhibition and disruption of the actin cytoskeleton by cytochalasin D. Both events are down-stream targets of Vav-1. Overall, our findings indicate that the TcR and TcR-CD28 driven PKB-GSK-3 pathway can operate independently of Vav-1 in T-cells.

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Disturbed B Cell Homeostasis in Newly Diagnosed Giant Cell Arteritis and Polymyalgia Rheumatica

Arthritis & Rheumatology ◽

10.1002/art.38625 ◽

2014 ◽

Vol 66 (7) ◽

pp. 1927-1938 ◽

Cited By ~ 57

Author(s):

Kornelis S. M. van der Geest ◽

Wayel H. Abdulahad ◽

Paulina Chalan ◽

Abraham Rutgers ◽

Gerda Horst ◽

...

Keyword(s):

B Cell ◽

Giant Cell Arteritis ◽

Giant Cell ◽

Polymyalgia Rheumatica ◽

Newly Diagnosed ◽

Cell Homeostasis ◽

B Cell Homeostasis

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Lymph node formation and B cell homeostasis require IKK-α in distinct endothelial cell–derived compartments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2100195118 ◽

2021 ◽

Vol 118 (48) ◽

pp. e2100195118

Author(s):

Kelly A. McCorkell ◽

Nipun Jayachandran ◽

Michelle D. Cully ◽

Jacquelyn Freund-Brown ◽

Tiffany Weinkopff ◽

...

Keyword(s):

Lymph Node ◽

B Cell ◽

Vascular Endothelial Cells ◽

Hematopoietic Cells ◽

Lymphatic Vessels ◽

Cell Homeostasis ◽

Cell Numbers ◽

Iκb Kinase ◽

Physiologic Function ◽

B Cell Homeostasis

Global inactivation of IκB kinase (IKK)-α results in defective lymph node (LN) formation and B cell maturation, and loss of IKK-α–dependent noncanonical NF-κB signaling in stromal organizer and hematopoietic cells is thought to underlie these distinct defects. We previously demonstrated that this pathway is also activated in vascular endothelial cells (ECs). To determine the physiologic function of EC-intrinsic IKK-α, we crossed IkkαF/F mice with Tie2-cre or Cdh5-cre mice to ablate IKK-α in ECs. Notably, the compound defects of global IKK-α inactivation were recapitulated in IkkαTie2 and IkkαCdh5 mice, as both lacked all LNs and mature follicular and marginal zone B cell numbers were markedly reduced. However, as Tie2-cre and Cdh5-cre are expressed in all ECs, including blood forming hemogenic ECs, IKK-α was also absent in hematopoietic cells (HC). To determine if loss of HC-intrinsic IKK-α affected LN development, we generated IkkαVav mice lacking IKK-α in only the hematopoietic compartment. While mature B cell numbers were significantly reduced in IkkαVav mice, LN formation was intact. As lymphatic vessels also arise during development from blood ECs, we generated IkkαLyve1 mice lacking IKK-α in lymphatic ECs (LECs) to determine if IKK-α in lymphatic vessels impacts LN development. Strikingly, while mature B cell numbers were normal, LNs were completely absent in IkkαLyve1 mice. Thus, our findings reveal that IKK-α in distinct EC-derived compartments is uniquely required to promote B cell homeostasis and LN development, and we establish that LEC-intrinsic IKK-α is absolutely essential for LN formation.

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Dual Phosphorylation of Btk by Akt/Protein Kinase B Provides Docking for 14-3-3ζ, Regulates Shuttling, and Attenuates both Tonic and Induced Signaling in B Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00247-13 ◽

2013 ◽

Vol 33 (16) ◽

pp. 3214-3226 ◽

Cited By ~ 26

Author(s):

Dara K. Mohammad ◽

Beston F. Nore ◽

Alamdar Hussain ◽

Manuela O. Gustafsson ◽

Abdalla J. Mohamed ◽

...

Keyword(s):

Protein Kinase ◽

B Cell ◽

Tyrosine Phosphorylation ◽

Nuclear Translocation ◽

Kinase Inhibitor ◽

Protein Kinase B ◽

Negative Regulator ◽

Pleckstrin Homology Domain ◽

Loss Of Function ◽

Interaction Sites

Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in theBtkgene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.

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