Serve H, Yee N S, Stella G, Sepp-Lorenzino L, Tan J C, Besmer P
Molecular Biology Program, Sloan Kettering Institute, New York, NY 10021.
EMBO J. 1995 Feb 1;14(3):473-83. doi: 10.1002/j.1460-2075.1995.tb07023.x.
The pleiotropic effects of the Kit receptor system are mediated by Kit-Ligand (KL) induced receptor autophosphorylation and its association with and activation of distinct second messengers, including phosphatidylinositol 3'-kinase (PI3-kinase), p21ras and mitogen-activated protein kinase (MAPK). To define the role of PI3-kinase, p21ras and MAPK in Kit-mediated cell proliferation, survival and adhesion in bone marrow-derived mast cells (BMMC), mutant Kit receptors were expressed in Wsh/Wsh BMMC lacking endogenous c-kit expression. The introduction of both murine Kit(S) and KitL (isoform containing a four amino acid insert) into Wsh/Wsh BMMC restored KL-induced proliferation, survival and adhesion to fibronectin, as well as activation of PI3-kinase, p21ras and MAPK, and induced expression of c-fos, junB, c-myc and c-myb mRNA. Substitution of tyrosine 719 in the kinase insert with phenylalanine (Y719F) abolished PI3-kinase activation, diminished c-fos and junB induction, and impaired KL-induced adhesion of BMMC to fibronectin. In addition, the Y719F mutation had partial effects on p21ras activation, cell proliferation and survival, while MAP kinase activation was not affected. On the other hand, Y821F substitution impaired proliferation and survival without affecting PI3-kinase, p21ras and MAPK activation, and induction of c-myc, c-myb, c-fos and c-jun mRNA, while KL-induced cell adhesion to fibronectin remained intact. In agreement with a role for PI3-kinase in Kit-mediated cell adhesion, wortmannin blocked Kit-mediated cell adhesion at concentrations known to specifically inhibit PI3-kinase. We conclude, that association of Kit with p85PI3-K, and thus with PI3-kinase activity, is necessary for a full mitogenic as well as adhesive response in mast cells. In contrast, tyrosine 821 is essential for Kit-mediated mitogenesis and survival, but not cell adhesion.
Kit受体系统的多效性作用是由Kit配体（KL）诱导的受体自身磷酸化及其与不同第二信使的结合和激活介导的，这些第二信使包括磷脂酰肌醇3'-激酶（PI3-激酶）、p21ras和丝裂原活化蛋白激酶（MAPK）。为了确定PI3-激酶、p21ras和MAPK在Kit介导的骨髓来源肥大细胞（BMMC）增殖、存活和黏附中的作用，在缺乏内源性c-kit表达的Wsh/Wsh BMMC中表达了突变型Kit受体。将小鼠Kit(S)和KitL（包含四个氨基酸插入片段的异构体）导入Wsh/Wsh BMMC可恢复KL诱导的增殖、存活以及与纤连蛋白的黏附，同时恢复PI3-激酶、p21ras和MAPK的激活，并诱导c-fos、junB、c-myc和c-myb mRNA的表达。将激酶插入片段中的酪氨酸719替换为苯丙氨酸（Y719F）可消除PI3-激酶的激活，减少c-fos和junB的诱导，并损害KL诱导的BMMC与纤连蛋白的黏附。此外，Y719F突变对p21ras激活、细胞增殖和存活有部分影响，而MAP激酶激活不受影响。另一方面，Y821F替换损害增殖和存活，但不影响PI3-激酶、p21ras和MAPK激活以及c-myc、c-myb、c-fos和c-jun mRNA的诱导，而KL诱导的细胞与纤连蛋白的黏附保持完整。与PI3-激酶在Kit介导的细胞黏附中的作用一致，渥曼青霉素在已知特异性抑制PI3-激酶的浓度下可阻断Kit介导的细胞黏附。我们得出结论，Kit与p85PI3-K的结合以及因此与PI3-激酶活性的结合对于肥大细胞的完全促有丝分裂以及黏附反应是必需的。相比之下，酪氨酸821对于Kit介导的有丝分裂和存活至关重要，但对细胞黏附并非必需。
