Seiji Inui

Alpha4 protein as a common regulator of type 2A‐related serine/threonine protein phosphatases1

The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits. It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A‐related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6. In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro. In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6. These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A‐related protein phosphatases.

Involvement of a rapamycin-sensitive pathway in CD40-mediated activation of murine B cells in vitro

Activation of resting B cells requires an initial triggering of the B cell antigen receptor (BCR) and secondary stimuli through various cytokine receptors and B cell activation molecules including CD40. We found that activation of B cells through CD40 is selectively inhibited by an immunosuppressant drug, rapamycin. This effect of rapamycin on anti-CD40-mediated activation of B cells was observed using three different in vitro assays. Rapamycin suppressed the anti-CD40-induced proliferation of splenic B cells, suppressed differentiation to surface IgMhigh/IgDlow B cells, and inhibited an anti-CD40-mediated prevention of apoptosis induced by BCR cross-linkage of WEHI-231 cells. We next examined several known CD40 signal transduction pathways to identify the target of rapamycin in stimulated B cells. Rapamycin did not inhibit the activation of c-Jun N-terminal kinases (JNKs) induced by anti-CD40 stimulation nor the activation of immediate nuclear transcription factors of NF-kappaB. Therefore, rapamycin affects a novel element of the CD40 signal transduction pathway which influences the proliferation, differentiation, and prevention of apoptosis of B cells.

Regulation of CaMKII by α4/PP2Ac contributes to learning and memory

Ca(2+)-dependent CaMKIIalpha activation with autophosphorylation plays an essential role in learning and memory. The regulation of CaMKIIalpha by dephosphorylation by protein phosphatase 1 (PP1) has been demonstrated. We addressed whether the protein phosphatase 2A (PP2A) that is abundant in the brain could be involved in the regulation of CaMKIIalpha. CaMKIIalpha was associated with the catalytic subunit of PP2A (PP2Ac) and alpha4, a regulator of PP2A. To investigate whether alpha4 plays an important role in the CNS, we established a neuron specific Cre transgenic mouse and a neuron specific alpha4 deficient mouse (N-alpha4 KO mouse). This N-alpha4 KO mouse showed impaired learning and memory in a water maze and also shuttle-box avoidance test. The activity of CaMKIIalpha also increased in hippocampus. An overexpression of alpha4 in the neuronal cell line demonstrated the activity of CaMKIIalpha to be regulated by alpha4. alpha4 and PP2Ac were localized in the cytoplasm but not in the postsynaptic density (PSD), thus suggesting that the dephosphorylation of CaMKIIalpha by alpha4/PP2Ac occurred in the cytoplasm. These results suggest that alpha4 and PP2A may thus play an important role in CaMKIIalpha regulation and thereby also influence learning.

T cell-specific gene targeting reveals that α4 is required for early T cell development

α4‐mediated signaling is involved in a variety of functions in mammalian cells. To determine whether this is true for immunocompetent cells, we generated mutant (Lck‐α4–) micein which the α4 gene was deleted in a T cell‐specific manner using the Cre/loxP system. These mice showed impaired early T cell development. Thymi at most ages were small and their architecturewas disorganized. This defect was not due to increased thymocyte apoptosis but to decreased cell proliferation. T cell development was found to be severely arrested at the CD4/CD8 double‐negative 3stage and the thymus contained very few double‐positive or single‐positive (SP) mature thymocytes. The mutant thymocytes showed impaired proliferative responses to anti‐CD3 monoclonal antibody (mAb) stimulation or to the cytokines IL‐2, IL‐1 or TNF. In the spleen, the numbers of mature SP T cells were decreased and their proliferative responses to anti‐CD3 plus IL‐2 or to anti‐CD28 mAb were impaired. A severe impairment of CD3‐induced expression of CD25 was also observed. These data suggest that α4 plays a critical role in the proliferation of thymocytes, which is necessary for early T cell development.

CaMKII phosphorylates serine 10 of p27 and confers apoptosis resistance to HeLa cells.

Protein phosphatase (PP) 6 is a serine threonine phosphatase which belongs to the PP2A subfamily of protein phosphatases. PP6 has been implicated in the control of apoptosis. A dominant negative form PP6 (DN-PP6) mutant cDNA was prepared and transfected into HeLa cells to investigate the regulation of apoptosis. HeLa cells expressing DN-PP6 showed increased resistance to apoptosis induced by TNF and cycloheximide. CaMKII phosphorylation and the expression of p27 were increased in DN-PP6 transfectants. Transient expression or activation of CaMKII increased the expression of p27. Furthermore, CaMKII phosphorylated serine 10 of p27, which induces the translocation of p27 from nucleus to cytoplasm and increases the stability of p27. Overexpression of wild type but not the S10A mutant p27 cDNA increased the expression of Bcl-xL and conferred apoptosis resistance to HeLa cells. These results indicated that PP6 and CaMKII regulated apoptosis by controlling the expression level of p27.

The origin of IgG-containing cells in the bursa of Fabricius

The bursa of Fabricius of the chicken is known as a primary lymphoid organ for B-cell development. Morphologically, the origin of IgG-containing cells in the bursa has not been clear until now, because abundant maternal IgG (MIgG) is transported to the chick embryo and distributed to the bursal tissue around hatching. Thus, it has been difficult to find out whether these cells themselves biosynthesize IgG or if they acquire MIgG via attachment to their surface. Our present study employing in situ hybridization clarified that IgG-containing cells in the medulla of bursal follicles did not biosynthesize IgG. To study the role of MIgG in the development of those IgG-containing cells, MIgG-free chicks were established from surgically bursectomized hen (SBx-hen). We found that, on the one hand, deprivation of MIgG from chicks completely inhibited the development of IgG-containing cells in the medulla after hatching. On the other hand, administration of MIgG to MIgG-free chicks recovered the emergence of those cells. In addition, we observed that those cells did not bear a B-cell marker and possessed dendrites with aggregated IgG. These results demonstrate that IgG-containing cells in the medulla are reticular cells that capture aggregated MIgG. Moreover, we show that the isolation of the bursa from environmental stimuli by bursal duct ligation (BDL) suppressed the development of IgG-containing cells after hatching. Thus, it is implied that environmental stimulations play a key role in MIgG aggregations and dendritic distributions of aggregated MIgG in the medulla after hatching.

IgE class switch recombination is regulated by CaMKII via NF-ΚB alternative pathway

IgE class switch recombination (CSR) is under the tight control to maintain the low concentration of serum IgE. IL-4 and CD40 signal induces IgE CSR. NF-κB classical pathway and STAT6 was indicated in this pathway. In this study, we prepared a M12 cell line expressing TRAF3 in an inducible manner. This cell line showed impaired signal transduction of NF-κB alternative pathway and diminished expression of epsilon germ line transcription (εGLT), thus indicating NF-κB alternative pathway was involved in IgE CSR. CaMKII was activated after IL-4 and anti-CD40 Ab stimulation, and KN-93, a CaMKII inhibitor, inhibited both activation of NF-κB alternative pathway and IgE CSR. NF-κB alternative pathway is characterized by the NIK activation after TRAF3 degradation through ubiquitination. This ubiquitination of TRAF3 was enhanced by CaMKII and alpha4 in HEK293T cells. Alpha4 was phosphorylated by CaMKII, and alpha4 associated with TRAF3. CaMKII also regulated IgE CSR in normal splenic B cells induced by IL-4 and anti-CD40 Ab stimulation. Introduction B cells develop in the bone marrow to produce IgM class antibody by the assembly of V, D and J region of Ig gene independently of the stimulation by antigens [1,2]. When mature B cells encounter with antigens in the peripheral tissue, they may produce different class of antibody with the same antigen specificity by class switch recombination (CSR) to acquire different types of effector functions [3,4]. CSR requires both CD40 signaling and stimulation by cytokines [5]. CD40 ligation is required in all CSR events and induces the activation of AID. AID acts on the single stranded DNA produced by the transcription of switch region [3,6]. Cytokines induce the germ line transcription (GLT) of switch region, thus determining the specificity of the class switch. IgE class antibody is related to the development of allergy and thus is under the control of tight regulation [7]. IL-4/IL-13 stimulation induce the ɛGLT and thereby IgE CSR [8,9]. Iɛ promoter region has STAT6 and NF-κB sites and it was reported that STAT6 and NF-κB classical pathway played an important role in the regulation of IgE CSR [10]. NF-κB is a group of transcription factors including NF-κB1 (p105/ p50), NF-kB2 (p100/p52), RelA (p65), RelB and c-Rel [11,12]. They reside in cytoplasm as inactive forms and translocate to nucleus when cells are activated. Various stimulations induce the processing of p105 to p50 and p100 to p52 [13]. This activation of NF-κB is mediated by classical and alternative pathways. Classical pathway is activated in many cells by various stimulations. On the other hand, alternative pathway is mainly activated in B cells by ligation of a limited number of receptors including TNF receptor family members such as CD40 and BAFFR [14,15]. The main transcription factors involved in alternative pathway are RelB and p52 of p100 [11]. The degradation of p100 to p52 is mediated by NIK [11]. NIK phosphorylates and activates IKKα, and then IKKα phosphorylates p100 to induce polyubiquitination and subsequent processing of p100 by proteasome. The amount of NIK protein is low in the non-stimulated cells, because E3 ligases cIAP1/2 ubiquitinate NIK for degradation with the help of TRAF3 [11,15,16]. When receptor is ligated by its ligand, cIAP1/2 ubiquitinate and degrade TRAF3 instead, by assembling to the receptor. TRAF3 belongs to TRAF family and regulates the NF-κB alternative pathway negatively [17,18]. TRAF3 associates with CD40, and the mutant form of CD40 that cannot bind to TRAF3 failed to induce IgE CSR [19]. Alpha4 was originally identified as an associated molecule of B cell receptor (BCR) complex [20]. Later studies demonstrated that alpha4 was ubiquitously expressed and was a regulator of serine/threonine phosphatases such as PP2A and PP6 [21,22]. Alpha4 is an essential molecule for cell survival and the deletion of alpha4 gene even in ES cells was lethal [23,24]. Conditional gene targeting of alpha4 in B cells revealed that alpha4 was involved not only in BCR signal transduction but also in many other signal pathways including LPS, CD40 and IL-4 receptor [25]. B cell specific alpha4 deficient mice showed decreased level of IgG subclasses upon immunization with T dependent Ag. The response of IgM class Ab was intact in the same mice, suggesting that CSR was impaired in alpha4 deficient mice [25]. Neuron specific gene targeting of alpha4 led to the finding that alpha4 regulated learning and memory by associating with CaMKIIα in the cytoplasm of neuron cells [26]. Correspondence to: Seiji Inui, MD, PhD Department of Immunology and Hematology, Faculty of Life Sciences, Graduate School of Health Sciences, Kumamoto University, 1-2-42, Kuhonji, Chuo-ku, Kumamoto, 862-0976, Japan, Tel; +81-96-373-5503, Fax; +81-96-356-3125; E-mail: inui@kumamoto-u.ac.jp