Kinase anchor protein muteins, peptides thereof and related methods

Abstract

A-kinase anchor protein (AKAPS) muteins, peptides thereof, and nucleic acids encoding the peptides are provided herein. Also provided are transgenic animals, cells comprising transgenes and various methods employing such peptides.

1 - 106 . (canceled) 
     
     
         107 . An isolated nucleic acid molecule, comprising a sequence of nucleotides that encodes a polypeptide that is a D-AKAP2 mutein, or a polypeptide fragment thereof, comprising a peptide region corresponding to an A-Kinase binding (AKB) domain set forth as amino acids 623-649 of SEQ ID NOs:64 or 65, wherein the sequence of the AKB domain peptide region corresponds to a peptide mutein that exhibits modified binding to a regulatory subunit of PKA compared to a native D-AKAP2. 
     
     
         108 . A nucleic acid vector, comprising the nucleic acid molecule of  claim 107 . 
     
     
         109 . A cell containing the nucleic acid vector of  claim 108 . 
     
     
         110 . A method of producing a D-AKAP2 mutein by growing the cell of  claim 109  under conditions whereby the D-AKAP2 mutein is expressed; and isolating the mutein. 
     
     
         111 . The method of  claim 110 , wherein the cell is a mammalian cell, yeast cell, insect cell or bacterial cell. 
     
     
         112 . The method of  claim 111 , wherein the mammalian cell is a human cell. 
     
     
         113 . The isolated nucleic acid molecule of  claim 107 , wherein the polypeptide is a mutein of SEQ ID NO: 2, and the polypeptide exhibits modified binding to a regulatory subunit of PKA compared to a peptide having SEQ ID NO: 2. 
     
     
         114 . The isolated nucleic acid molecule of  claim 113 , wherein the polypeptide is selected from the group consisting of
 a) a peptide having the sequence SEQ ID NO: 2;   b) a peptide having the sequence SEQ ID NO: 2 further comprising a C-terminal cysteine;   c) a peptide having the sequence SEQ ID NO: 2 wherein residues 1-8 are deleted; and   d) a peptide having the sequence SEQ ID NO: 2 wherein residues 1-8 are deleted further comprising a C-terminal cysteine;   except that said peptide has an amino acid residue substitution at the residue equivalent to residue 21 of SEQ ID NO: 2 of Trp or Ile for Val, and/or an amino acid residue substitution at the residue equivalent to residue 12 of SEQ ID NO: 2 of Phe for Leu; and   zero, one, or two amino acid residue substitutions selected from the group consisting of Phe, Ile, Leu, Val, His, Met, Arg, Thr, Trp, or Tyr at the residue equivalent to residue 9 of SEQ ID NO: 2 for Gln; and Phe, Ile, Leu, Thr, Val, Trp, or Tyr at the residue equivalent to residue 25 of SEQ ID NO: 2 for Met.   
     
     
         115 . The isolated nucleic acid molecule of  claim 107 , wherein the polypeptide exhibits enhanced binding to PKA-RIα subunits. 
     
     
         116 . The isolated nucleic acid molecule of  claim 107 , wherein the polypeptide exhibits normal or reduced binding to PKA-RIIα subunits. 
     
     
         117 . The isolated nucleic acid molecule of  claim 107 , wherein the polypeptide exhibits normal or increased binding to PKA-RIα subunits. 
     
     
         118 . The isolated nucleic acid molecule of  claim 107 , wherein the polypeptide exhibits a preferred or exclusive binding to PKA-RIα subunits relative to PKA-RIIα subunits. 
     
     
         119 . The isolated nucleic acid molecule of  claim 107 , wherein the polypeptide has enhanced ability to bind to PKA-RIα subunit, and a reduced ability to bind to PKA-RIIα subunit, compared to the peptide of SEQ ID NOs:1 or 2, wherein the peptide is selected from the group consisting of: 
       
         
           
                 
                 
                 
               
                   FEELAWKIAKMIWSDVMQQC; 
                   (SEQ ID NO:104; PV-37) 
                     
                 
                     
                 
                   FEELAWKIAKMIWSDVFQQC; 
                   (SEQ ID NO:103; PV-38) 
                 
                     
                 
                   QEEFAWKIAKMIVSDVFQQC; 
                   (SEQ ID NO:105; PV-47) 
                 
                     
                 
                   QEEFAWKIAKMIISDVFQQC;. 
                   (SEQ ID NO:106; PV-48) 
                 
             
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         120 . The isolated nucleic acid molecule of  claim 114 , wherein the polypeptide comprises an amino acid substitution at residue 21 of Ile or Trp for Val. 
     
     
         121 . The isolated nucleic acid molecule of  claim 114 , wherein the polypeptide comprises an amino acid substitution at residue 21 of Trp for Val. 
     
     
         122 . The isolated nucleic acid molecule of  claim 120 , wherein the polypeptide comprises an amino acid substitution at residue 9 of Phe for Gln. 
     
     
         123 . The isolated nucleic acid molecule of  claim 107 , wherein the polypeptide is a peptide having the sequence SEQ ID NO: 2 wherein amino acids 1-8 are deleted or a peptide having the sequence SEQ ID NO: 2 wherein amino acids 1-8 are deleted further comprising a C-terminal cysteine. 
     
     
         124 . The isolated nucleic acid molecule of  claim 120 , wherein the polypeptide is a peptide having the sequence SEQ ID NO: 2 wherein amino acids 1-8 are deleted or a peptide having the sequence SEQ ID NO: 2 wherein amino acids 1-8 are deleted further comprising a C-terminal cysteine. 
     
     
         125 . The isolated nucleic acid molecule of  claim 121 , wherein the polypeptide is a peptide having the sequence SEQ ID NO: 2 wherein amino acids 1-8 are deleted or a peptide having the sequence SEQ ID NO: 2 wherein amino acids 1-8 are deleted further comprising a C-terminal cysteine. 
     
     
         126 . The isolated nucleic acid molecule of  claim 107 , wherein the polypeptide binds to the PKA-RIα subunit but has substantially no ability to bind to the PKA-RIIα subunit, compared to the peptide of SEQ ID NOs:1 or 2, wherein the peptide is selected from the group consisting of: 
       
         
           
                 
                 
                 
               
                   VQGNTDEAQEEFAWKIAKMIVSD[I/V]MQQ; 
                   (SEQ ID NO:51) 
                     
                 
                     
                 
                   VQGNTDEAQEELAWKIAKMIISD[I/V]MQQ; 
                   (SEQ ID NO:52) 
                 
                   and 
                 
                     
                 
                   VQGNTDEAQEELAWKIAKMILSD[I/V]MQQ.. 
                   (SEQ ID NO:53) 
                 
             
                
                
                
                
                
                
               
            
           
         
       
     
     
         127 . The isolated nucleic acid molecule of  claim 107 , wherein the polypeptide has an enhanced ability to bind to PKA-RI RIα subunit, and a reduced ability to bind to PKA-RIIα subunit, compared to the peptide of SEQ ID NOs:1 or 2, wherein the peptide is VQGNTDEAQEELAWKIAKMIWSD[I/V]MQQ (SEQ ID NO:54). 
     
     
         128 . The isolated nucleic acid molecule of  claim 107 , wherein the polypeptide is FEELAWKIAKMIISDVFQQC (SEQ ID NO:107; PV-49).