Identification and analysis of tyrosine phosphatase CD45 interacting proteins

CD 45和白细胞共同抗原相关蛋白（LAR）与大多数跨膜蛋白酪氨酸磷酸酶（RTPases）一样，在胞浆段中具有两个串联重复的酪氨酸磷酸酶结构域。从共表达的CD 3 zeta、lck和CD 45 D1CS（其828位的半胱氨酸被丝氨酸取代）的裂解物中，使用抗CD 45抗体共沉淀磷酸化的CD 3 zeta和CD 45 D1CS。这一发现表明CD 3 zeta在体内也是CD 45的良好底物。制备了三种CD 3 zeta突变体MC 1、MC 2、MC 3，它们分别含有C端第2和第3、第1和第3、第1和第2个酪氨酸残基。3种突变体均能被Ick磷酸化，但与CD 45 D1CS的亲和力几乎相同，而SHC-SH 2不能与磷酸化的MC 2结合; CD 45 D1 D2CS的1144位半胱氨酸被丝氨酸取代，与CD 3 zeta的亲和力较低，与磷酸化的CD 3 zeta的亲和力较CD 45 D1CS低。并研究了LAR与胰岛素受体（IR）的相互作用。LAR与胰岛素刺激后酪氨酸磷酸化的胰岛素受体相关并优先去磷酸化。LARD 1CS能稳定结合磷酸化IR。IR可能是LAR的生理底物，在胰岛素刺激后，LAR被IR激酶磷酸化。LARD 1D 2CS结构域1813位半胱氨酸被丝氨酸取代，与IR的亲和力较LARD ICS低，磷酸化程度较高，提示LARD 2具有自脱磷酸化活性。这些结果表明，CD 45和LAR的D1 PTP具有主要的磷酸酶活性，而D2 PTP可以独立于磷酸化而识别底物。

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.