Yasuhisa Fukui

Novel Role of Phosphatidylinositol 3-Kinase in CD28-mediated Costimulation

Ligation of the CD28 surface receptor provides a major costimulatory signal for full scale T cell activation. Despite extensive studies, the intracellular signaling pathways delivered by CD28 ligation are not fully understood. A particularly controversial matter is the role of phosphatidylinositol 3-kinase (PI3K) in CD28-mediated costimulation. It is known that the binding site for PI3K and Grb-2 lies nested within the YMNM motif of the CD28 cytoplasmic domain. To elucidate the role of PI3K during CD28-mediated interleukin-2 (IL-2) production, CD28 YMNM point and deletion mutants were expressed in Jurkat cells. We then measured IL-2 promoter activation after CD28 ligation. Our results showed that the Y189F mutant, which disrupts binding by PI3K, and the YMNM deletion mutant both demonstrated reduced but significant activity for IL-2 promoter activation. In contrast, the N191A mutant, which retains PI3K binding ability, resulted in a complete abrogation of activity, suggesting that PI3K mediates a negative effect upon transcriptional activation of theIL-2 gene. Consistent with this idea, we found that the addition of a PI3K pharmacological inhibitor augmented IL-2 promoter activity, whereas coexpression of a constitutively active form of PI3K reduced this activity. Taken together, these data indicate that PI3K, when associated with the YMNM motif, may act as a negative mediator in CD28-mediated IL-2 gene transcription.

Membrane recruitment of DOCK180 by binding to PtdIns(3,4,5)P3.

DOCK180 was originally identified as one of two major proteins bound to the Crk oncogene product and became an archetype of the CDM family of proteins, including Ced-5 of Caenorhabditis elegans and Mbc of Drosophila melanogaster. Further study has suggested that DOCK180 is involved in the activation of Rac by the CrkII-p130(Cas) complex. With the use of deletion mutants of DOCK180, we found that the C-terminal region containing a cluster of basic amino acids was required for binding to and activation of Rac. This region showed high amino-acid sequence similarity to the consensus sequence of the phosphoinositide-binding site; this led us to examine whether this basic region binds to phosphoinositides. For this purpose we used PtdIns(3,4,5)P(3)-APB beads, as reported previously [Shirai, Tanaka, Terada, Sawada, Shirai, Hashimoto, Nagata, Iwamatsu, Okawa, Li et al. (1998) Biochim. Biophys. Acta 1402, 292-302]. By using various competitors, we demonstrated the specific binding of DOCK180 to PtdIns(3,4,5)P(3). The expression of active phosphoinositide 3-kinase (PI-3K) did not enhance a DOCK180-induced increase in GTP-Rac; however, the expression of PI-3K translocated DOCK180 to the plasma membrane. Thus DOCK180 contained a phosphoinositide-binding domain, as did the other guanine nucleotide exchange factors with a Dbl homology domain, and was translocated to the plasma membrane on the activation of PI-3K.

Activation of ErbB3-PI3-kinase pathway is correlated with malignant phenotypes of adenocarcinomas.

Signet-ring cell carcinomas are malignant dedifferentiated carcinomas, which are frequently found in the stomach. We previously demonstrated that a 200u2009kDa protein is often constitutively phosphorylated on tyrosine and bound to phosphatidylinositol 3-kinase (PI3-kinase) in signet-ring cell carcinoma cells. In this study, we purified the 200u2009kDa protein from an extract of NUGC-4 cells, a cell line of signet-ring cell carcinoma, and identified it as ErbB3. ErbB3 was found to be phosphorylated selectively in dedifferentiated adenocarcinoma cell lines among various gastric cancer cell lines. Expression of a constitutively active chimeric receptor consisting of ErbB2 and ErbB3 in HCC2998 cells, a highly differentiated adenocarcinoma cell line, revealed that the signaling triggered by phosphorylation of ErbB3 was important for dedifferentiated phenotypes such as loss of cell–cell interaction and high expression of MUC1/DF3 antigen, a marker of the malignant tumors. Taken together, activation of ErbB3 pathway may contribute to the development of dedifferentiated carcinomas.

Role of phosphatidylinositol 3-phosphate in formation of forespore membrane in Schizosaccharomyces pombe

Phosphatidylinositol (PI) 3‐kinase (encoded by the pik3+ gene) in Schizosaccharomyces pombe has been identified as a homologue of VPS34p, a protein required for proper vesicular protein sorting. The clone defective in this protein carries enlarged vacuoles and exhibits sensitivity to high temperature or high ion concentration. The effect of disruption of pik3+ on sporulation of Sz. pombe was examined. The diploid cells underwent G1 arrest and meiosis. However, the spores formed by the Δpik3 cells were not viable. Electron‐microscopic analysis revealed that the growth of the forespore membrane of Δpik3 cells was not correctly orientated, failing to engulf the nucleus or forming extremely small spores, as was confirmed by the use of Spo3p–GFP and GFP–Psy1p, which are markers of the forespore membrane. The coating materials found along the forespore membrane of the wild‐type were greatly reduced in these cells. PI 3‐P, the product of Pik3p, was detected on the forespore membrane, suggesting that PI 3‐P‐dependent vesicle transport may take place in formation of the forespore membrane. Misshaped forespore membrane, accumulation of vesicles, formation of small non‐viable spores, and suppression by over expression of Psy1p were the phenotypes commonly seen in Δpik3 and Δspo3 cells, suggesting a relationship between the functions of Pik3p and Spo3p in formation of the forespore membrane in Sz. pombe. Copyright

Sorting nexin homologues are targets of phosphatidylinositol 3-phosphate in sporulation of Schizosaccharomyces pombe

Schizosaccharomyces pombe defective in phosphatidylinositol (PtdIns) 3‐kinase shows various defects in forespore membrane formation, including onset, growth orientation, and closure. Downstream factors of PtdIns 3‐kinase in this system were explored. Among various phox homology (PX) domain‐containing proteins, Vps5p and Vps17p, homologues of sorting nexins, were found to be required for efficient sporulation. Cells defective in these proteins showed a disordered growth orientation of the forespore membrane, as is the case with Δpik3 cells. Vps5p and Vps17p with mutations in the PX domains failed to suppress the defects of their relevant disruptants. Vps5p and Vps17p migrated toward the the forespore membrane in a pik3+‐dependent manner, suggesting that these proteins may interact with PtdIns(3)P. Electron‐microscopic analysis revealed that the forespore membrane fails to engulf the nucleus in some of these cells, accumulating vesicle‐like bodies similar to those seen in Δspo3 cells. These results suggest that Vps5p and Vps17p are the targets of PtdIns(3)P in vesicle transport required for onset of the forespore membrane formation.

The PI 3-kinase-Rac-p38 MAP kinase pathway is involved in the formation of signet-ring cell carcinoma

Signet-ring cell carcinoma is classified in poorly differentiated adenocarcinoma with an aggressive nature and a poor prognosis. We have shown that the activation of PI 3-kinase in highly differentiated adenocarcinomas induces loss of cell–cell contact and formation of vacuoles, giving phenotypes similar to those of signet-ring cell lines. SB203580, a potent p38 MAP kinase inhibitor, blocked this transition, and expression of an active form of MKK6 (MKK6DA), an activator of p38 MAP kinase, gave effects similar to those induced by expression of the active form of PI 3-kinase (BD110), although formation of large vacuoles was not induced. Activation of MKK3, another activator of p38 MAP kinase, was activated in native signet-ring carcinoma cell lines. Anchorage-independent growth of signet-ring cell lines was inhibited by LY294002 or SB203580. These results suggest that p38 MAP kinase is functioning downstream of PI 3-kinase in signaling of the malignant phenotype. Secretion of mucins was enhanced in BD110-expressing cells, but not in MKK6DA-expressing cells, suggesting that secretion of mucins is independent of the MKK6-p38 MAP kinase cascade. Thus, there may be at least two pathways, p38 MAP kinase-dependent and -independent, which are involved in regulation of cell–cell contact and the protein secretion system, respectively.

Inportance of Phosphatidylinositol 3-Phosphate in Sporulation of Schizosaccharomyces pombe

In Schizosaccharomyces pombe, Pik3p phosphorylates phosphatidylinositol (PI) to produce PI 3-P, which is further phosphorylated by Ste12p to yield PI 3,5-P2. Pik3p is required for both conjugation and sporulation. To test which of PI 3-P and PI 3,5-P2 is required for sporulation, diploid cells defective in production of PI 3,5-P2 were used. They underwent sporulation almost normally provided that the osmotic pressure of the medium was controlled, suggesting that not PI 3,5-P2 but PI 3-P was important. Electron microscopic analysis confirmed normal sporulation in the absence of PI 3,5-P2 although the forespore membrane was found to be less dense in these cells.