Coding sequence for protein phosphatase methylesterase, recombinant DNA molecules and methods

Abstract

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 carboxy-terminus by specific methylesterase. Carboxymethylation affects PP2A activity and varies during the cell cycle. The present disclosure provides the coding sequence of a methylesterase, herein named Protein Phosphatase Methylesterase-1 (PME-1). PME-1 is highly conserved from yeast to human and contains a motif found in lipases, which motif has a catalytic triad-activated serine as the active site nucleophile. Recombinant PME-1 polypeptide produced in bacteria demethylates PP2A C subunit in vitro and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. PME-1 represents the first mammalian protein phosphatase methylesterase cloned to date.

We claim:  
     
         1 . An isolated nucleic acid molecule comprising a portion encoding protein phosphatase methylesterase-1 (PME-1), wherein said portion comprises a sequence at least 70% identical to a nucleotide sequence given in SEQ ID NO:9, nucleotides 1-1200.  
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein said molecule encodes a PME-1 polypeptide consisting essentially of an amino acid sequence as given in SEQ ID NO:6, amino acids 1-400.  
     
     
         3 . The nucleic acid molecule of  claim 2 , wherein said molecule comprises a PME-1 coding sequence as shown in SEQ ID NO:9, nucleotide 1-1200.  
     
     
         4 . A recombinant expression vector comprising the nucleic acid molecule encoding protein phosphatase methylesterase-1 (PME-1) of  claim 1 , wherein a coding sequence of said molecule is operably linked to and expressed under control of transcription and translation regulatory elements.  
     
     
         5 . The expression vector of  claim 4 , wherein the encoded PME-1 consists essentially of the amino acid sequence given in SEQ ID NO:6, amino acids 1-400.  
     
     
         6 . The expression vector of  claim 4 , wherein said vector comprises the PME-1 coding sequence substantially as given in SEQ ID NO:9, nucleotides 1-1200.  
     
     
         7 . The expression vector of  claim 4 , wherein said vector is a bacterial vector.  
     
     
         8 . The expression vector of  claim 4 , wherein said vector is a baculovirus vector.  
     
     
         9 . The expression vector of  claim 4 , wherein said vector is a mammalian vector.  
     
     
         10 . A recombinant host cell, wherein said cell comprises the expression vector of  claim 4 .  
     
     
         11 . The host cell of  claim 10 , wherein said cell is a recombinant bacterial cell.  
     
     
         12 . The host cell of  claim 10 , where said cell is a recombinant mammalian cell.  
     
     
         13 . A method for producing a recombinant protein phosphatase methylesterase-1 (PME-1) polypeptide, said method comprising the steps of: 
 (a) introducing the expression vector of  claim 4  into a host selected from the group consisting of bacteria, insects and mammals; and    (b) culturing under conditions where PME-1 polypeptide is produced,    whereby said PME-1 polypeptide shows protein phosphatase methylsterase activity in vitro.    
     
     
         14 . An isolated polypeptide or fragment thereof, having protein phosphatase methylesterase-1 activity, the amino acid sequence of which comprises residues 1-400 of SEQ ID NO: 6.