Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned. Carboxymethylation affects PP2A activity and varies during the cell cycle. Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins. One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned. PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile. Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned. Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.Protein phosphatase 2A (PP2A) 1 is a highly conserved serine/threonine phosphatase involved in the regulation of a wide variety of enzymes, signal transduction pathways, and cellular events (1, 2). Consonant with its diverse roles, subpopulations of PP2A have been found to localize to the nucleus, cytoplasm, cytoskeleton, and membranes (3-6). The smallest functional unit of PP2A thought to exist in vivo consists of a heterodimer between a catalytic 36-kDa subunit, termed C, and a constant regulatory 63-kDa subunit, termed A (7). This A/C heterodimer often further complexes with a member of one of three additional cellular regulatory subunit families termed B (or B55), BЈ (or B56), and BЉ (or PR72/120) (1). In cells stably transformed by the middle tumor antigen (MT) of polyomavirus, MT substitutes for the B subunit in a small portion (ϳ10%) (8) 2 of PP2A complexes (9). MT⅐PP2A complex formation is known to be important for MT-mediated transformation (10 -13), but the precise functional consequences of MT association with PP2A are still being elucidated.Efforts aimed at understanding PP2A regulation have uncovered a complex set of noncovalent and covalent mechanisms. These include association with different regulatory subunits (1), association with heat stable inhibitors (14), action of a phosphotyrosyl activator protein (15), lipid binding (16), phosphorylation (17), and methylation (18 -22). These mechanisms affect the catalytic activity, substrate specificity, and cellular localization of PP2A. However, little is known about the molecular bases of their effects, and even less about how th...

Interaction between the heterodimeric form of protein phosphatase 2A (PP2A) and polyomavirus middle T antigen (MT) is required for the subsequent assembly of a transformation-competent MT complex. To investigate the role of PP2A catalytic activity in MT complex formation, we undertook a mutational analysis of the PP2A 36-kDa catalytic C subunit. Several residues likely to be involved in the dephosphorylation mechanism were identified and mutated. The resultant catalytically inactive C subunit mutants were then analyzed for their ability to associate with a cellular (B subunit) or a viral (MT) B-type subunit. Strikingly, while all of the inactive mutants were severely impaired in their interaction with B subunit, most of these mutants formed complexes with polyomavirus MT. These findings indicate a potential role for these catalytically important residues in complex formation with cellular B subunit, but not in complex formation with MT. Transformation-competent MT is known to associate with, and modulate the activity of, several cellular proteins, including pp60c-src family kinases. To determine whether association of MT with an active PP2A A-C heterodimer is necessary for subsequent association with pp60c-src , catalytically inactive C subunits were examined for their ability to form complexes containing pp60c-src in MT-expressing cells. Two catalytically inactive C subunit mutants that efficiently formed complexes with MT also formed complexes that included an active pp60c-src kinase, demonstrating that PP2A activity is not essential in cis in MT complexes for subsequent pp60c-src association.

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Denaturing gradient gel electrophoresis (DGGE) is commonly used to search for point mutations in DNA fragments amplified in vitro by the polymerase chain reaction (PCR). For the complete detection of mutations in large genes with many exons, the DGGE‐PCR approach, or any other PCR‐based method, requires many primer sets and amplification reactions to scan the entire protein‐coding sequence. We previously demonstrated that DGGE analysis using DNA blots detects mutations in Drosophila genes and sequence polymorphisms in human genes without prior PCR amplification. To determine if human point mutations could be detected using denaturing gradient gels (DGG blots), genomic DNA samples from hemophilia A families were analyzed for mutations in the factor VIII (FVIII) gene. Restriction enzyme digested DNA samples were subjected to DGGE and transferred to nylon blots. Hybridization of the DGG blots with FVIII cDNA probes revealed mutant and polymorphic DNA sequence differences. Among 26 affected families that were not carriers of intron 22 inversion mutations, 18 family‐specific DNA fragment polymorphisms and one multiexon deletion were mapped. DNA sequencing of eight patient‐specific polymorphic DNA fragments revealed six single base change mutations, one 4 bp deletion, and one 13 bp duplication. Hum Mutat 12:393–402, 1998. © 1998 Wiley‐Liss, Inc.