Cloning and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to retinaldehyde-binding protein and yeast SEC14p.

Protein tyrosine phosphorylation is important in the regulation of cell growth, the cell cycle, and malignant transformation. We have cloned a cDNA that encodes a cytosolic protein-tyrosine-phosphatase (PTPase), MEG2, from MEG-01 cell and human umbilical vein endothelial cell cDNA libraries. The 4-kilobase cDNA sequence of PTPase MEG2 corresponds in length to the mRNA transcript detected by Northern blotting. The predicted open reading frame encodes a 68-kDa protein composed of 593 amino acids and has no apparent signal or transmembrane sequences, suggesting that it is a cytosolic protein. The C-terminal region has a PTPase catalytic domain that has 30-40% amino acid identity to other known PTPases. The N-terminal region has 254 amino acids that are 28% identical to cellular retinaldehyde-binding protein and 24% identical to yeast SEC14p, a protein that has phosphatidylinositol transfer activity and is required for protein secretion through the Golgi complex in yeast. Recombinant PTPase MEG2 expressed in Escherichia coli possesses PTPase activity. PTPase MEG2 mRNA was detected in 12 cell lines tested, which suggests that this phosphatase is widely expressed. The structure of PTPase MEG2 implies that a tyrosine phosphatase could participate in the transfer of hydrophobic ligands or in functions of the Golgi apparatus. IMAGES:

蛋白质酪氨酸磷酸化（Protein tyrosine phosphorylation）在细胞生长、细胞周期及恶性转化的调控过程中发挥关键作用。我们从MEG-01细胞与人类脐静脉内皮细胞的互补DNA（cDNA）文库中，克隆得到了编码胞质蛋白酪氨酸磷酸酶（cytosolic protein-tyrosine-phosphatase, PTPase）MEG2的cDNA。该PTPase MEG2的4kb cDNA序列长度与通过Northern印迹（Northern blotting）检测到的mRNA转录本一致。预测的开放阅读框（open reading frame）编码一个由593个氨基酸残基组成的68kDa蛋白质，无明显信号肽或跨膜序列，提示其为胞质蛋白。其C端区域含有PTPase催化结构域，与其他已知PTPase的氨基酸序列同源性达30%~40%。N端区域的254个氨基酸残基，与细胞视黄醛结合蛋白（cellular retinaldehyde-binding protein）的同源性为28%，与酵母SEC14p的同源性为24%；SEC14p是一种具有磷脂酰肌醇转移活性的蛋白质，且在酵母中参与经高尔基体（Golgi complex）的蛋白质分泌过程。在大肠杆菌（Escherichia coli）中表达的重组PTPase MEG2具备PTPase活性。在本次检测的12种细胞系中均能检测到PTPase MEG2的mRNA，提示该磷酸酶的表达具有广谱性。PTPase MEG2的结构特征提示，酪氨酸磷酸酶可能参与疏水配体的转运或高尔基体的功能调控。图像：