Biomedical subjects

Yasuyuki Kitaura

Publications and source records attributed to Yasuyuki Kitaura.

6 recordsLinked to original sources

PubMed2006

Establishment of the major compatibility complex-dependent development of CD4+ and CD8+ T cells by the Cbl family proteins.

Casitas B cell lymphoma (Cbl) proteins are negative regulators for T cell antigen receptor (TCR) signaling. Their role in thymocyte development remains unclear. Here we show that simultaneous inactivation of c-Cbl and Cbl-b in thymocytes enhanced thymic negative selection and altered the ratio of CD4(+) and CD8(+) T cells. Strikingly, the mutant thymocytes developed into CD4(+)- and CD8(+)-lineage T cells independent of the major histocompatibility complex (MHC), indicating that the CD4(+)- and CD8(+)-lineage development programs are constitutively active in the absence of c-Cbl and Cbl-b. The mutant double-positive (DP) thymocytes exhibited spontaneous hyperactivation of nuclear factor-kappa B (NF-kappaB). Additionally, they failed to downregulate the pre-TCR and pre-TCR signaling. Thus, our data indicate that Cbl proteins play a critical role in establishing the MHC-dependent CD4(+) and CD8(+) T cell development programs. They likely do so by suppressing MHC-independent NF-kappaB activation, possibly through downmodulating pre-TCR signaling in DP thymocytes.

Adaptor Proteins, Signal Transducing↗

PubMed2005

PKA-mediated phosphorylation regulates the function of activation-induced deaminase (AID) in B cells.

During humoral immune responses, two distinct genetic modification events diversify the Ig genes in germinal center (GC) B cells: somatic hypermutation and class switch recombination (CSR). Both processes require the activity of activation-induced cytidine deaminase (AID), an enzyme expressed specifically in GC B cells. However, the mechanisms that regulate AID activity are largely unknown. Here we report that protein kinase A (PKA) phosphorylates AID and regulates its activity in GC B cells. AID physically interacts with the PKA holoenzyme in the cytoplasm and is phosphorylated by the PKA catalytic subunit at specific residues. AID phosphorylation is required for CSR, because substitution of the two phosphorylation targets impairs its ability to rescue CSR in AID-deficient B cells. Pharmacologic inhibition of PKA prevents isotype class switching in a murine B-cell lymphoma cell line; conversely, B cells from mice where PKA activity is made constitutive by conditional deletion of the PKA regulatory subunit gene display enhanced CSR. These findings implicate PKA in the regulation of AID function and suggest that the control of T cell-dependent immune responses may be modulated, via AID, by signals that activate PKA.

Amino Acid Sequence↗

PubMed2004

The penta-EF-hand protein ALG-2 interacts with a region containing PxY repeats in Alix/AIP1, which is required for the subcellular punctate distribution of the amino-terminal truncation form of Alix/AIP1.

ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family and associates with several proteins, including annexin VII, annexin XI, and Alix/AIP1, in a Ca(2+)-dependent manner. The yeast two-hybrid system and a biotin-tagged ALG-2 overlay assay were carried out to characterize the interaction between ALG-2 and Alix. The region corresponding to amino acid residues 794 to 827 in the carboxy-terminal proline-rich region of Alix was sufficient to confer the ability to interact directly with ALG-2. This region includes four-tandem PxY repeats. Alanine substitutions indicated that seven proline residues in this region, four in the PxY repeats, and four tyrosine residues in the PxY repeats are crucial for the binding affinity with ALG-2. Endogenous ALG-2 was co-immunoprecipitated in the presence of Ca(2+) with FLAG-tagged Alix or FLAG-tagged Alix Delta EBS, a deletion mutant lacking the endophilin binding consensus sequence, but not with FLAG-tagged Alix Delta ABS, another mutant lacking the region comprising amino acids 798-841, from the lysates of HEK293 cells transfected with each FLAG-tagged protein expression construct. FLAG-tagged ALG-2 overexpressed in HEK293 cells was also co-immunoprecipitated with Alix in a Ca(2+)-dependent fashion, whereas FLAG-tagged ALG-2(E47A/E114A), a Ca(2+)-binding deficient mutant of ALG-2, was not detected in the immunoprecipitates of Alix even in the presence of Ca(2+). Fluorescent microscopic analyses using the carboxy-terminal half of Alix fused with green fluorescent protein (GFP-AlixCT) revealed that endogenous ALG-2 in HeLa cells exhibits a dot-like pattern overlapping with exogenously expressed GFP-AlixCT, and the distribution of GFP-AlixCT Delta ABS is observed diffusely in the cytoplasm. These results indicate the requirement of ABS in Alix for the efficient accumulation of AlixCT and raise the possibility that ALG-2 participates in membrane trafficking through a Ca(2+)-dependent interaction with Alix.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Structures, functions and molecular evolution of the penta-EF-hand Ca2+-binding proteins.

Penta-EF-hand (PEF) proteins comprise a family of Ca(2+)-binding proteins that have five repetitive EF-hand motifs. Among the eight alpha-helices (alpha1-alpha8), alpha4 and alpha7 link EF2-EF3 and EF4-EF5, respectively. In addition to the structural similarities in the EF-hand regions, the PEF protein family members have common features: (i) dimerization through unpaired C-terminal EF5s, (ii) possession of hydrophobic Gly/Pro-rich N-terminal domains, and (iii) Ca(2+)-dependent translocation to membranes. Based on comparison of amino acid sequences, mammalian PEF proteins are classified into two groups: Group I PEF proteins (ALG-2 and peflin) and Group II PEF proteins (Ca(2+)-dependent protease calpain subfamily members, sorcin and grancalcin). The Group I genes have also been found in lower animals, plants, fungi and protists. Recent findings of specific interacting proteins have started to gradually unveil the functions of the noncatalytic mammalian PEF proteins.

Amino Acid Sequence↗

PubMed2002

ALG-2 interacts with the amino-terminal domain of annexin XI in a Ca(2+)-dependent manner.

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family. ALG-2 forms a homodimer, a heterodimer with another penta-EF-hand protein, peflin, and a complex with its interacting protein, named AIP1 or Alix. By yeast two-hybrid screening using human ALG-2 as bait, we isolated a cDNA of a novel ALG-2-interacting protein, which turned out to be annexin XI. Deletion analysis revealed that ALG-2 interacted with the N-terminal domain of annexin XI (AnxN), which has an amino acid sequence similar to that of the C-terminal region of AIP1/Alix. Using recombinant biotin-tagged ALG-2 and the glutathione S-transferase (GST) fusion protein of AnxN, the direct interaction was analyzed by an ALG-2 overlay assay and by real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. The dissociation constant (K(d)) was estimated to be approximately 70 nM. The Ca(2+)-dependent fluorescence change of ALG-2 in the presence of the hydrophobicity fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was inhibited by mixing with GST-AnxN, suggesting that the Pro/Gly/Tyr/Ala-rich hydrophobic region in AnxN masked the Ca(2+)-dependently exposed hydrophobic surface of ALG-2.

Amino Acid Sequence↗

PubMed2002

Both ALG-2 and peflin, penta-EF-hand (PEF) proteins, are stabilized by dimerization through their fifth EF-hand regions.

ALG-2 (apoptosis-linked gene-2 protein) and peflin are Ca(2+)-binding proteins and belong to the penta-EF-hand (PEF) protein family, which includes calpain, sorcin, and grancalcin. ALG-2 forms either a homodimer or a heterodimer with peflin like other PEF proteins. In this study, we found that the fifth-EF-hand (EF-5) regions of both ALG-2 and peflin are essential for dimerization and their stabilities. Exogenously expressed EF-5-deletion (DeltaEF-5) mutants of ALG-2 and peflin were unstable and were not detected in HEK293 cells by Western blotting. In a pulse--chase experiment, the DeltaEF-5 mutants were rapidly degraded, but they were stabilized by treatment with a proteasome inhibitor, MG132. In MG132-treated cells, DeltaEF-5 mutants were recovered in the insoluble fractions. Transient coexpression of ALG-2 increased the peflin level. These results indicate that the absence of a fifth EF-hand results in rapid degradation by the proteasome. On the other hand, stable expression of exogenous peflin decreased the amount of endogenous peflin. The amount of peflin that can dimerize with ALG-2 seems to be restricted in mammalian cells.

Apoptosis Regulatory Proteins↗