The effect of overexpression of the protein tyrosine phosphatase PTPMEG on cell growth and on colony formation in soft agar in COS-7 cells
The effect of overexpression of the protein tyrosine phosphatase PTPMEG on cell growth and on colony formation in soft agar in COS-7 cells
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DOI：
10.1073/pnas.93.23.12980
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发表时间：
1996-11-12
影响因子：
11.1
通讯作者：
Majerus, PW
中科院分区：
文献类型：
--
作者：
Gu, MX;Meng, K;Majerus, PW
我们建立了稳定高表达重组PTPMEG的COS-7细胞系和活性部位半胱氨酸突变为丝氨酸的失活突变形式(PTPMEGCS)。我们发现，内源性和重组酶都主要定位于COS-7细胞的膜和细胞骨架部分。内源性PTPMEG仅占COS-7细胞总酪氨酸磷酸酶活性的1/3000，而转基因细胞表达的酪氨酸磷酸酶水平是COS-7细胞的2-7倍。这些过表达水平没有导致总酪氨酸磷酸酶活性或蛋白酪氨酸磷酸化状态的检测到的变化，这是通过用抗磷酸酪氨酸抗体对细胞匀浆进行免疫印迹来确定的。尽管PTPMEG的活性水平较低，但我们发现过度表达的细胞生长较慢，并以较低的密度达到融合。令人惊讶的是，尽管PTPMEGCS细胞的生长密度高于PTPMEG细胞，但PTPMEGCS转基因细胞的融合密度也低于载体转染细胞。两种构建物都能抑制COS-7细胞在软琼脂中形成集落的能力，其中天然PTPMEG的作用(30倍)大于PTPMEGCS(10倍)。这些结果表明，在COS-7细胞中，PTPMEG和PTPMEGCS都抑制了细胞的增殖，降低了细胞的饱和密度，并阻止了这些细胞在没有附着固体基质的情况下生长的能力。
We established stable COS-7 cell lines overexpressing recombinant PTPMEG and an inactive mutant form in which the active site cysteine is mutated to serine (PTPMEGCS). We found that both endogenous and recombinant enzyme were primarily located in the membrane and cytoskeletal fractions of COS-7 cells. Endogenous PTPMEG accounts far only 1/3000th of the total tyrosine phosphatase activity in COS-7 cells and transfected cells expressed 2- to 7-fold higher levels of the enzyme. These levels of overexpression did not result in detectable changes in either total tyrosine phosphatase activity or the state of protein tyrosine phosphorylation as determined by immunoblotting of cell homogenates with anti-phosphotyrosine antibodies. Despite the low levels of activity for PTPMEG, we found that overexpressing cells grew slower and reached confluence at a lower density than vector transfected cells. Surprisingly, PTPMEGCS-transfected cells also reach confluence at a lower density than vector-transfected cells, although they grow to higher density than PTPMEG-transfected cells. Both constructs inhibited the ability of COS-7 cells to form colonies in soft agar, with the native PTPMEG having a greater effect (30-fold) than PTPMEGCS (10-fold). These results indicate that in COS-7 cells both PTPMEG and PTPMEGCS inhibit cell proliferation, reduce the saturation density, and block the ability of these cells to grow without adhering to a solid matrix.