Del Vecchio R L, Tonks N K
Cold Spring Harbor Laboratory, New York 11724-2208.
J Biol Chem. 1994 Jul 29;269(30):19639-45.
We have chosen Xenopus laevis as a model system to study how protein tyrosine phosphatases (PTPases) function in growth and development. As an initial step, we have previously isolated in a polymerase chain reaction (PCR)-based protocol cDNA fragments which correspond to sequences within the catalytic domains of PTPases (Yang, Q., and Tonks, N. K. (1993) Adv. Protein Phosphatases 7, 359-372). Two of these PCR products, designated X1 and X10, have now been used to screen a X. laevis ovary cDNA library to obtain complete coding sequences for two distinct PTPases. The X1 cDNA encodes a protein (PTPX1) of 693 amino acids (approximately 79 kDa); the X10 cDNA encodes a protein of 597 amino acids (approximately 69 kDa). Both PTPX1 and PTPX10 lack potential membrane spanning sequences and therefore can be classified as non-transmembrane/cytoplasmic PTPases. While the overall structure of these PTPases are similar, sharing segments of 95% amino acid identity, they differ in that PTPX1 contains a unique 97-amino acid insert between the N-terminal segment and C-terminal catalytic domain. The absence of complete identity between PTPX1 and PTPX10 suggests that these two sequences are the products of separate genes and not the result of alternative splicing. This conclusion is confirmed by PCR analysis of Xenopus genomic DNA. Both PTPases share sequence identities in their N-terminal segments with two lipid-binding proteins, cellular retinaldehyde-binding protein and SEC14p, a phospholipid transferase. In addition, the unique insert sequence of PTPX1 shares identity with PSSA, a protein involved in phosphatidylserine biosynthesis. Sequence comparison suggests that PTPX10 is the Xenopus homolog of the human PTPase Meg-02, while PTPX1 is a structurally related yet distinct PTPase. Intrinsic PTPase activity of PTPX1 and PTPX10 was demonstrated in lysates of Sf9 cells infected with recombinant baculoviruses encoding either enzyme. PTPX1 can be recovered in both soluble and membrane fractions from Xenopus oocytes with the membrane form exhibiting approximately 4-fold higher activity than the soluble form.
我们选择非洲爪蟾作为模型系统，以研究蛋白质酪氨酸磷酸酶（PTPases）在生长和发育过程中的功能。作为第一步，我们之前通过基于聚合酶链反应（PCR）的方法分离出了与PTPases催化结构域内序列相对应的cDNA片段（Yang, Q., 和Tonks, N. K. (1993) Adv. Protein Phosphatases 7, 359 - 372）。现在，这两个PCR产物，分别命名为X1和X10，已被用于筛选非洲爪蟾卵巢cDNA文库，以获得两种不同PTPases的完整编码序列。X1 cDNA编码一个由693个氨基酸组成的蛋白质（PTPX1，约79 kDa）；X10 cDNA编码一个由597个氨基酸组成的蛋白质（约69 kDa）。PTPX1和PTPX10都缺乏潜在的跨膜序列，因此可归类为非跨膜/细胞质PTPases。虽然这些PTPases的整体结构相似，氨基酸序列有95%的同源性，但它们的不同之处在于，PTPX1在N端和C端催化结构域之间有一个独特的97个氨基酸的插入序列。PTPX1和PTPX10之间不存在完全相同的序列，这表明这两个序列是不同基因的产物，而不是可变剪接的结果。非洲爪蟾基因组DNA的PCR分析证实了这一结论。这两种PTPases在其N端序列上与两种脂质结合蛋白，即细胞视黄醛结合蛋白和磷脂转移酶SEC14p，具有序列同源性。此外，PTPX1独特的插入序列与参与磷脂酰丝氨酸生物合成的蛋白质PSSA具有同源性。序列比较表明，PTPX10是人类PTPase Meg - 02的非洲爪蟾同源物，而PTPX1是一种结构相关但不同 的PTPase。在用编码这两种酶的重组杆状病毒感染的Sf9细胞裂解物中，证实了PTPX1和PTPX10的内在PTPase活性。PTPX1可以从非洲爪蟾卵母细胞的可溶性和膜部分中回收，膜形式的活性比可溶性形式高约4倍。
