title: N-(cyclohexanecarboxyl)-O-phospho-l-serine, a minimal substrate for the dual-specificity protein phosphatase IphP.
abstract: Three dual-specific phosphatases [DSPs], IphP, VHR, and Cdc14, and three protein-tyrosine phosphatases [PTPs], PTP-1B, PTP-H1, and Tc-PTPa, were challenged with a set of low molecular weight phosphoesters to probe the factors underlying the distinct substrate specificities displayed by these two mechanistically homologous families of protein phosphatases. It was observed that beta-naphthyl phosphate represented an excellent general substrate for both PTPs and DSPs. While DSPs tended to hydrolyze alpha-naphthyl phosphate at rates comparable to that of the beta-isomer, the PTPs PTP-1B and Tc-PTPa did not. PTP-H1, however, displayed high alpha-naphthyl phosphatase activity. Intriguingly, PTP-H1 also displayed much higher protein-serine phosphatase activity in vitro, 0.2-0.3% that toward equivalent tyrosine phosphorylated proteins, than did PTP-1B or Tc-PTPa. The latter two PTPs discriminated between the serine- and tyrosine-phosphorylated forms of two test proteins by factors of >/=10(4)-10(6). While free phosphoserine represented an extremely poor substrate for all of the DSPs examined, the addition of a hydrophobic "handle" to form N-(cyclohexanecarboxyl)-O-phospho-l-serine produced a compound that was hydrolyzed by IphP with high efficiency, i.e., at a rate comparable to that of free phosphotyrosine or p-nitrophenyl phosphate. VHR also hydrolyzed N-(cyclohexanecarboxyl)-O-phospho-l-serine (1 mM) at a rate approximately one-tenth that of beta-naphthyl phosphate. None of the PTPs tested exhibited significant activity against this compound. However, N-(cyclohexanecarboxyl)-O-phospho-l-serine did not prove to be a universal substrate for DSPs as Cdc14 displayed little propensity to hydrolyze it.
authors.affiliation: Department of Chemistry, Institute for Genomics, Blacksburg, Virginia 24061, USA.
authors.forename: P S
authors.initials: PS
authors.lastname: Savle
authors.forename: T E
authors.initials: TE
authors.lastname: Shelton
authors.forename: C A
authors.initials: CA
authors.lastname: Meadows
authors.forename: M
authors.initials: M
authors.lastname: Potts
authors.forename: R D
authors.initials: RD
authors.lastname: Gandour
authors.forename: P J
authors.initials: PJ
authors.lastname: Kennelly
mesh_terms: D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology; D003510:Cyclohexanes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D054640:Dual Specificity Phosphatase 3; D006801:Humans; D006868:Hydrolysis; D007536:Isomerism; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D009113:Muramidase / Q000378:metabolism; D004676:Myelin Basic Protein / Q000378:metabolism; D009281:Naphthalenes / Q000737:chemistry / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010768:Phosphoserine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C074860:CDC14 protein, S cerevisiae; D018797:Cell Cycle Proteins; D003510:Cyclohexanes; D009097:Multienzyme Complexes; D004676:Myelin Basic Protein; C407063:N-(cyclohexanecarboxyl)-O-phospho-serine; C407064:N-benzoyl-O-phospho-serine; D009281:Naphthalenes; D009943:Organophosphorus Compounds; D029701:Saccharomyces cerevisiae Proteins; D010768:Phosphoserine; D019000:Phosphotyrosine; C014694:naphthyl phosphate; D012694:Serine; C080609:protein-tyrosine phosphatase, Nostoc; C517025:DUSP3 protein, human; D054640:Dual Specificity Phosphatase 3; D017027:Protein Tyrosine Phosphatases; D009113:Muramidase