Functional Analysis of Conserved Domains in the Phosphotyrosyl Phosphatase Activator. Molecular Cloning of the Homologues from Drosophila melanogaster and Saccharomyces cerevisiae † , ‡
University of Leuven, Louvain, Flemish, Belgium Biochemistry
(Impact Factor: 3.02).
10/1998; 37(37):12899-908. DOI: 10.1021/bi980496l
Phosphotyrosyl phosphatase activator (PTPA), a 37 kDa cytosolic protein that specifically activates the phosphotyrosyl phosphatase activity of the dimeric form of PP2A, was cloned from Drosophila melanogaster and Saccharomyces cerevisiae. Sequence alignment of PTPA from yeast to human revealed highly conserved regions including the type B fragment of the putative PTPA ATP binding site. We generated PTPA deletion mutants of these conserved regions as well as point mutations within regions that were suggested to be functionally important. The recombinant proteins were expressed in E. coli and subsequently purified. Activity measurements, linked with immunological detection, revealed that most of the well-conserved regions are essential for PTPA activity. However, neither the type A fragment of the putative ATP binding site nor the cysteine-rich region, present in all but the Drosophila and yeast homologues, appeared to be essential for PTPA activity. Moreover, we observed that PTPA truncated at glycine266 behaves as a dominant negative mutant since it is inhibitory to the wild-type PTPA.
Available from: Sophie Quevillon-Cheruel
- "In previous investigations, we showed that the conserved region 200 GVWGLD 205 of PTPA (Ypa1 numbering) is essential for both the activation of the tyrosyl phosphatase activity of PP2A D as well as for the activation of PP2A i and the PPIase activity (Jordens et al., 2006; Van Hoof et al., 1998). Deletion of this region diminished PTPA activity. "
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ABSTRACT: PTPA, an essential and specific activator of protein phosphatase 2A (PP2A), functions as a peptidyl prolyl isomerase (PPIase). We present here the crystal structures of human PTPA and of the two yeast orthologs (Ypa1 and Ypa2), revealing an all alpha-helical protein fold that is radically different from other PPIases. The protein is organized into two domains separated by a groove lined by highly conserved residues. To understand the molecular mechanism of PTPA activity, Ypa1 was cocrystallized with a proline-containing PPIase peptide substrate. In the complex, the peptide binds at the interface of a peptide-induced dimer interface. Conserved residues of the interdomain groove contribute to the peptide binding site and dimer interface. Structure-guided mutational studies showed that in vivo PTPA activity is influenced by mutations on the surface of the peptide binding pocket, the same mutations that also influenced the in vitro activation of PP2Ai and PPIase activity.
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Available from: Veerle Janssens
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ABSTRACT: The phosphotyrosine phosphatase activator (PTPA) has been isolated as an in vitro regulator of protein phosphatase 2A. Human PTPA is encoded by a single gene, the structure and chromosomal localization of which have been determined in our previous work. Here we describe the further isolation, sequencing and functional characterization of the PTPA promoter region. In agreement with its ubiquitous expression, the PTPA promoter displays several characteristics of housekeeping genes: it lacks both a TATA-box and a CAAT-box, it is very GC-rich and it contains an unmethylated CpG island surrounding the transcription initiation site. Transient transfection experiments in different cell types with several truncated chimaeric luciferase reporter gene plasmids revealed the importance of the region between positions -67 and -39 for basal promoter activity. This region coincides remarkably well with the determined CpG island. Further analysis of this region demonstrated the presence of a Yin Yang 1 (YY1) binding motif at positions -52 to -44. Binding of YY1 to this sequence is demonstrated in bandshift and DNase I footprinting experiments. Another YY1 binding motif is found in the 5' untranslated region, at positions +27 to +35. Mutations in either of these sites, abolishing YY1 binding in vitro, have differential effects on promoter activity. Point mutations in both sites completely abolish promoter activity. Moreover, induction of promoter activity by co-transfection with a YY1 expression plasmid is fully dependent upon the presence of both intact YY1 binding sites. Thus YY1 apparently mediates basal transcription of the human PTPA gene through two binding sites within its proximal promoter.
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