The roles of cys124 and ser239 in the functional properties of human betaIII tubulin.
Tubulin is the target for some very powerful anti-mitotic and anti-tumor drugs. The betaIII tubulin isotype is found in very few normal tissues, but is often found in tumors, where it has been implicated in resistance to anti-tumor drugs. The betaIII isotype occurs in fish, amphibians, birds and mammals and its unique features are highly conserved in evolution. One of these features is the replacement of cys239 by ser239. Cys239 is unusual in being highly sensitive to oxidation; in fact, oxidation of this residue inhibits microtubule assembly. The betaIII isotype also has a very unusual cys124, where other beta isotypes have ser/ala124. The striking conservation in betaIII of vertebrates strongly suggests that cys124 and ser239 play functional roles. We have prepared the C124S and S239C mutants of betaIII and tested their effects on the functional properties of tubulin. We have found that both the betaIII C124S and betaIII S239C mutants bind colchicine less well than does wild-type alphabetaIII, and also make transfected HeLa cells more resistant to colchicine. However, the double mutant, betaIII C124S/S239C, binds colchicine still less well than do either of the single mutants, but in contrast to the former, the double mutant increases the cells' sensitivity to colchicine. Our results indicate that the roles that these residues play in colchicine binding and microtubule integrity are far more complex than previously imagined and that the specific residues at which betaIII differs from the other isotypes act collectively to keep betaIII in a functional conformation.
Joe
Patrick A
PA
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Banerjee
Asok
A
Ludueña
Richard F
RF
D000595:Amino Acid Sequence; D003078:Colchicine / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry / Q000502:physiology*; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
D050505:Mutant Proteins; D020033:Protein Isoforms; C498585:TUBB3 protein, human; D014404:Tubulin; D012694:Serine; D003545:Cysteine; D003078:Colchicine