Hisaaki Shinohara

B Cell Signaling and Fate Decision

Antigen receptors on the surface of B lymphocytes trigger adaptive immune responses after encountering their cognate antigens but also control a series of antigen-independent checkpoints during B cell development. These physiological processes are regulated by the expression and function of cell surface receptors, intracellular signaling molecules, and transcription factors. The function of these proteins can be altered by a dynamic array of post-translational modifications, using two interconnected mechanisms. These modifications can directly induce an altered conformational state in the protein target of the modification itself. In addition, they can create new binding sites for other protein partners, thereby contributing to where and when such multiple protein assemblies are activated within cells. As a new type of post-transcriptional regulator, microRNAs have emerged to influence the development and function of B cells by affecting the expression of target mRNAs.

Phospholipase C-γ2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen

B cell receptor (BCR) recognition of membrane-bound antigen initiates a spreading and contraction response, the extent of which is controlled through the formation of signaling-active BCR-antigen microclusters and ultimately affects the outcome of B cell activation. We followed a genetic approach to define the molecular requirements of BCR-induced spreading and microcluster formation. We identify a key role for phospholipase C-γ2 (PLCγ2), Vav, B cell linker, and Brutons tyrosine kinase in the formation of highly coordinated “microsignalosomes,” the efficient assembly of which is absolutely dependent on Lyn and Syk. Using total internal reflection fluorescence microscopy, we examine at high resolution the recruitment of PLCγ2 and Vav to microsignalosomes, establishing a novel synergistic relationship between the two. Thus, we demonstrate the importance of cooperation between components of the microsignalosome in the amplification of signaling and propagation of B cell spreading, which is critical for appropriate B cell activation.

PKCβ regulates BCR-mediated IKK activation by facilitating the interaction between TAK1 and CARMA1

The B cell antigen receptor (BCR)–mediated activation of IκB kinase (IKK) and nuclear factor–κB require protein kinase C (PKC)β; however, the mechanism by which PKCβ regulates IKK is unclear. Here, we demonstrate that another protein kinase, TGFβ-activated kinase (TAK)1, is essential for IKK activation in response to BCR stimulation. TAK1 interacts with the phosphorylated CARMA1 (also known as caspase recruitment domain [CARD]11, Bimp3) and this interaction is mediated by PKCβ. IKK is also recruited to the CARMA1–Bcl10–mucosal-associated lymphoid tissue 1 adaptor complex in a PKCβ-dependent manner. Hence, our data suggest that phosphorylation of CARMA1, mediated by PKCβ, brings two key protein kinases, TAK1 and IKK, into close proximity, thereby allowing TAK1 to phosphorylate IKK.

Role of Dok-1 and Dok-2 in myeloid homeostasis and suppression of leukemia

Dok-1 and Dok-2 are closely related rasGAP-associated docking proteins expressed preferentially in hematopoietic cells. Although they are phosphorylated upon activation of many protein tyrosine kinases (PTKs), including those coupled with cytokine receptors and oncogenic PTKs like Bcr-Abl, their physiological roles are largely unidentified. Here, we generated mice lacking Dok-1 and/or Dok-2, which included the double-deficient mice succumbed to myeloproliferative disease resembling human chronic myelogenous leukemia (CML) and chronic myelomonocytic leukemia. The double-deficient mice displayed medullary and extramedullary hyperplasia of granulocyte/macrophage progenitors with leukemic potential, and their myeloid cells showed hyperproliferation and hypo-apoptosis upon treatment and deprivation of cytokines, respectively. Consistently, the mutant myeloid cells showed enhanced Erk and Akt activation upon cytokine stimulation. Moreover, loss of Dok-1 and/or Dok-2 induced blastic transformation of chronic phase CML-like disease in mice carrying the bcr-abl gene, a cause of CML. These findings demonstrate that Dok-1 and Dok-2 are key negative regulators of cytokine responses and are essential for myeloid homeostasis and suppression of leukemia.

IκB kinase β–induced phosphorylation of CARMA1 contributes to CARMA1–Bcl10–MALT1 complex formation in B cells

Protein kinase C (PKC) β has been reported (Shinohara, H., T. Yasuda, Y. Aiba, H. Sanjo, M. Hamadate, H. Watarai, H. Sakurai, and T. Kurosaki. 2005. J. Exp. Med. 202:1423–1431; Sommer, K., B. Guo, J.L. Pomerantz, A.D. Bandaranayake, M.E. Moreno-Garcia, Y.L. Ovechkina, and D.J. Rawlings. 2005. Immunity. 23:561–574) to play a crucial role in B cell receptor (BCR)–mediated IκB kinase (IKK) activation through phosphorylation of caspase recruitment domain 11, Bimp3 (CARMA1). However, it remains unclear whether this PKCβ-mediated phosphorylation accounts fully for the activation status of CARMA1, because involvement of other kinases, such as phosphoinositide 3-kinase–dependent kinase 1, has also been suggested. We show that PKCβ mediates phosphorylation of CARMA1 on Ser668, which in turn is essential for BCR-mediated CARMA1–Bcl10–mucosal-associated lymphoid tissue 1 (MALT1) association and subsequent IKK activation. Our analyses also demonstrate that the downstream kinase IKKβ contributes to facilitating formation of the complex CARMA1–Bcl10–MALT1 by mediating phosphorylation of CARMA1. Hence, our data suggest that PKCβ is crucial for initial activation of IKK. The activated IKKβ does not merely function as an effector enzyme but also modifies the upstream signaling complex through a feedback mechanism, thereby optimizing the strength and duration of the nuclear factor κB signal.

Dok-1 and Dok-2 are negative regulators of lipopolysaccharide-induced signaling

Endotoxin, a bacterial lipopolysaccharide (LPS), causes fatal septic shock via Toll-like receptor (TLR)4 on effector cells of innate immunity like macrophages, where it activates nuclear factor κB (NF-κB) and mitogen-activated protein (MAP) kinases to induce proinflammatory cytokines such as tumor necrosis factor (TNF)-α. Dok-1 and Dok-2 are adaptor proteins that negatively regulate Ras–Erk signaling downstream of protein tyrosine kinases (PTKs). Here, we demonstrate that LPS rapidly induced the tyrosine phosphorylation and adaptor function of these proteins. The stimulation with LPS of macrophages from mice lacking Dok-1 or Dok-2 induced elevated Erk activation, but not the other MAP kinases or NF-κB, resulting in hyperproduction of TNF-α and nitric oxide. Furthermore, the mutant mice showed hyperproduction of TNF-α and hypersensitivity to LPS. However, macrophages from these mutant mice reacted normally to other pathogenic molecules, CpG oligodeoxynucleotides, poly(I:C) ribonucleotides, or Pam3CSK4 lipopeptide, which activated cognate TLRs but induced no tyrosine phosphorylation of Dok-1 or Dok-2. Forced expression of either adaptor, but not a mutant having a Tyr/Phe substitution, in macrophages inhibited LPS-induced Erk activation and TNF-α production. Thus, Dok-1 and Dok-2 are essential negative regulators downstream of TLR4, implying a novel PTK-dependent pathway in innate immunity.

Positive Feedback Within a Kinase Signaling Complex Functions as a Switch Mechanism for NF-κB Activation

Signaling Dynamics The signaling pathways that activate the transcription factor NF-κB are key regulatory pathways in cells of the immune system, and their dynamic properties are still being elucidated. In B cells, analysis of single-cell responses has shown that the stimulation of the B cell receptor causes a “digital” all-or-none response of cells to a stimulus. Shinohara et al. (p. 760) used a combination of mathematical modeling and experiments to show that this property of the system results from the presence of a positive feedback loop among the signaling components activated in response to the receptor. Studies in cells expressing mutated signaling components resolved key phosphorylation events that provide the threshold responses observed and identified potential molecular modifications that might modify the threshold or other aspects of the dynamic response. The molecular basis of an all-or-none response in B cells is revealed. A switchlike response in nuclear factor–κB (NF-κB) activity implies the existence of a threshold in the NF-κB signaling module. We show that the CARD-containing MAGUK protein 1 (CARMA1, also called CARD11)–TAK1 (MAP3K7)–inhibitor of NF-κB (IκB) kinase-β (IKKβ) module is a switch mechanism for NF-κB activation in B cell receptor (BCR) signaling. Experimental and mathematical modeling analyses showed that IKK activity is regulated by positive feedback from IKKβ to TAK1, generating a steep dose response to BCR stimulation. Mutation of the scaffolding protein CARMA1 at serine-578, an IKKβ target, abrogated not only late TAK1 activity, but also the switchlike activation of NF-κB in single cells, suggesting that phosphorylation of this residue accounts for the feedback.

Comprehending the complex connection between PKCβ, TAK1, and IKK in BCR signaling

Summary:  The transcription factor nuclear factor‐κB (NF‐κB) contributes to many events in the immune system. Characterization of NF‐κB has facilitated our understanding of immune cell differentiation, survival, proliferation, and effector functions. Intense research continues to elucidate the role of NF‐κB, which is shared in several receptor signaling pathways, such as Toll‐like receptors, the tumor necrosis factor receptor, and antigen receptors. The specificity of cellular responses emanating from stimulation of these receptors is determined by post‐translational modification, or ‘fine tuning’, which regulates spatiotemporal dynamics of downstream signaling. Understanding the fine tuning mechanisms of NF‐κB activation is crucial for insights into biological regulation and for understanding how cellular signaling pathways are tightly regulated to guide different cell fates. In this review, we focus on recent advances that illuminate the fine tuning mechanisms of NF‐κB activation by BCR signaling and have increased our comprehension of complex signal systems.

Dephosphorylation of Carma1 by PP2A negatively regulates T‐cell activation

The Carma1–Bcl10–Malt1 (CBM) complex bridges T‐cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF‐κB pathway. NF‐κB activation is triggered by PKCθ‐dependent phosphorylation of Carma1 after TCR/CD28 co‐stimulation. PKCθ‐phosphorylated Carma1 was suggested to function as a molecular scaffold that recruits preassembled Bcl10–Malt1 complexes to the membrane. We have identified the serine–threonine protein phosphatase PP2A regulatory subunit Aα (PPP2R1A) as a novel interaction partner of Carma1. PPP2R1A is associated with Carma1 in resting as well as activated T cells in the context of the active CBM complex. By siRNA‐mediated knockdown and in vitro dephosphorylation, we demonstrate that PP2A removes PKCθ‐dependent phosphorylation of Ser645 in Carma1, and show that maintenance of this phosphorylation is correlated with increased T‐cell activation. As a result of PP2A inactivation, we find that enhanced Carma1 S645 phosphorylation augments CBM complex formation, NF‐κB activation and IL‐2 or IFN‐γ production after stimulation of Jurkat T cells or murine Th1 cells. Thus, our data define PP2A‐mediated dephosphorylation of Carma1 as a critical step to limit T‐cell activation and effector cytokine production.

Dok-1 tyrosine residues at 336 and 340 are essential for the negative regulation of Ras-Erk signalling, but dispensable for rasGAP-binding

Dok‐1 is a common substrate of many protein tyrosine kinases (PTKs). It recruits rasGAP and other SH2‐containing proteins and negatively regulates Ras‐Erk signalling downstream of PTKs. However, the mechanisms of its inhibitory effect are yet unclear. Here, a series of C‐terminal deletion mutants of Dok‐1 delineated the core domain for the inhibition of Erk from 334 to 346 amino acid, which contains two SH2‐binding motifs having Tyr‐336 or Tyr‐340. The Dok‐1 mutants having tyrosine‐to‐phenylalanine (YF) substitution(s) at Tyr‐336 and/or Tyr‐340 lost their inhibitory effect on Ras and Erk downstream of Src‐like PTK, Lyn or Fyn, whereas the rasGAP‐binding of each mutant remained intact. However, the Dok‐1 mutant having YF substitutions at the rasGAP‐binding sites (Tyr‐295 and Tyr‐361) also showed incapability of Ras and Erk inhibition. Moreover, the Dok‐1 mutant having YF substitutions at Tyr‐336 and Tyr‐340 showed an impaired inhibitory effect on v‐Abl‐induced transformation of NIH‐3T3 cells. These results demonstrate that Tyr‐336 and Tyr‐340 of Dok‐1 are dispensable for rasGAP‐binding but essential for inhibition of Ras‐Erk signalling and cellular transformation downstream of PTKs. Thus, Dok‐1 probably recruits as yet unidentified molecule(s), which, in concert with rasGAP, negatively regulate Ras‐Erk signalling.