| Budget Amount *help |
¥18,060,000 (Direct Cost: ¥15,000,000、Indirect Cost: ¥3,060,000)
Fiscal Year 2009: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Fiscal Year 2008: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000)
Fiscal Year 2007: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2006: ¥4,800,000 (Direct Cost: ¥4,800,000)
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| Research Abstract |
Growth-blocking peptide (GBP) is an insect cytokine that stimulates a class of immune cells called plasmatocytes to adhere to each other and a foreign surface. Although structure-activity studies have been extensively performed in GBP and its mutants of Lepidoptera Pseudaletia separata, the signaling pathway of GBP-dependent activation of plasmatocytes remains unknown. We identified a novel adaptor protein (P77) with a molecular mass of 77kDa containing SH2/SH3 domain binding motifs and an immunoreceptor tyrosine-based activation motif (ITAM)-like domain in the cytoplasmic region of the C-terminus. Although P77 did not have the capacity for direct binding with GBP, its cytoplasmic tyrosine residues were specifically phosphorylated within seconds after addition of GBP to a plasmatocyte suspension. Tyrosine phosphorylation of P77 was also observed when hemocytes were incubated with Enterobactor cloacae or Micrococcus luteus, but this phosphorylation was found to be induced by GBP released from hemocytes stimulated by the pathogens. We further detected tyrosine phosphorylation of the integrin β subunit in plasmatocytes stimulated by GBP. Double-stranded RNAs targeting P77 not only decreased GBP-dependent tyrosine phosphorylation of the integrin β subunit, but also abolished the GBP-induced spreading of plasmatocytes on foreign surfaces. P77 RNAi larvae also showed significantly higher mortality than control larvae following infection with Serratia marcescens, thus indicating that P77 is essential for GBP to mediate a normal innate cellular immunity in insects. These results demonstrated that GBP signaling in plasmatocytes requires the adaptor protein P77 and that active P77-assisted tyrosine phosphorylation of integrins is critical for the activation of plasmatocytes.
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