CHARACTERIZATION OF 2 STRUCTURALLY RELATED XENOPUS-LAEVIS PROTEIN-TYROSINE PHOSPHATASES WITH HOMOLOGY TO LIPID-BINDING PROTEINS

We have chosen Xenopus laevis as a model system to study how protein t yrosine phosphatases (PTPases) function in growth and development. As an initial step, we have previously isolated in a polymerase chain rea ction (PCR)-based protocol cDNA fragments which correspond to sequence s within the catalytic domains of PTPases (Yang, and., and Tonks, N. K . (1993) Adv. Protein Phosphatases 7, 359-372). Two of these PCR produ cts, designated X1 and X10, have now been used to screen a X. laevis o vary cDNA library to obtain complete coding sequences for two distinct PTPases. The X1 cDNA encodes a protein (PTPX1) of 693 amino acids (ap proximate to 79 kDa); the X10 cDNA encodes a protein of 597 amino acid s (approximate to 69 kDa). Both PTPX1 and PTPX10 lack potential membra ne spanning sequences and therefore can be classified as non-transmemb rane/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-te rminal segment and C-terminal catalytic domain. The absence of complet e identity between PTPX1 and PTPX10 suggests that these two sequences are the products of separate genes and not the result of alternative s plicing. This conclusion is confirmed by PCR analysis of Xenopus genom ic DNA. Both PTPases share sequence identities in their N-terminal seg ments with two lipid-binding proteins, cellular retinaldehyde-binding protein and SEC14p, a phospholipid transferase. In addition, the uniqu e insert sequence of PTPX1 shares identity with PSSA, a protein involv ed in phosphatidylserine biosynthesis. Sequence comparison suggests th at PTPX10 is the Xenopus homolog of the human PTPase Meg-02, while PTP X1 is a structurally related yet distinct PTPase, Intrinsic PTPase act ivity of PTPX1 and PTPX10 was demonstrated in lysates of Sf9 cells inf ected with recombinant baculoviruses encoding either enzyme. PTPX1 can be recovered in both soluble and membrane fractions from Xenopus oocy tes with the membrane form exhibiting approximate to 4-fold higher act ivity than the soluble form.