| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
CD45 and leukocyte common antigen-related protein(LAR) have two tandem-repeated PTPase domains in the cytoplasmic segment like most of other transmembrane protein tyrosine phosphatases (RTPases). PTPase activity is associated only with the first of the two domains, PTPase domain 1(D1), and the membrane-distal PTPase domain 2(D2), which has no catalytic activity, would regulate substrate specificity.
From the lysate of co-expression of CD3zeta, lck and CD45D1CS, whose cystein at 828 has replaced with serine, phosphorylated CD3zeta was coprecipitated with CD45D1CS using anti-CD45 antibody. This finding indicates CD3zeta is a good substrate of CD45 also in vivo. Three kinds mutants MC1, MC2, MC3 of CD3zeta, which are carrying only 2 tyrosine residues, 2nd and 3rd, 1st and 3rd, 1st and 2nd tyrosine from C terminus, were made. All 3 mutants can be poorly phosphorylated with Ick and has almost same affinty with CD45D1CS, but SHC-SH2 can not bind with phosphorylated MC2 mutant.
CD45D1D2CS, whose cystein at 1144 was replaced with serine, has lower affinity with CD3zeta, and phsophorylated CD3zeta than CD45D1CS.Cystein of D2 has an important role of recognition of CD3zeta, independently of phosphorylation.
We studied on LAR and insuline receptor (IR) interaction also. LAR was associated with and preferentially dephosphorylated the insulin receptor which was tyrosine-phosphorylated after insulin stimulation. LARD1CS can bind phosphorylated IR stably. IR can be a phsiological substrate of LAR.LAR is phsophorylated with IRkinase after insuline stimulation. LARD1D2CS , whose cystein 1813 is changed to serine in the domain 2, has lower affinity with IR and higher phosphrylation than LARDICS.
These findings suggests LARD2 has auto-dephosphorylation activity. These data indicate that D1PTP of CD45 and LAR has main phosphatase activity and D2 PTP of them can recognize the substrates independently of phosphorylation.
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