Huntingtin phosphorylation sites mapped by mass spectrometry. Modulation of cleavage and toxicity.
Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing full-length myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development.
Schilling
Birgit
B
The Buck Institute for Age Research, Novato, California 94945, USA.
Gafni
Juliette
J
Torcassi
Cameron
C
Cong
Xin
X
Row
Richard H
RH
LaFevre-Bernt
Michelle A
MA
Cusack
Michael P
MP
Ratovitski
Tamara
T
Hirschhorn
Ricky
R
Ross
Christopher A
CA
Gibson
Bradford W
BW
Ellerby
Lisa M
LM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000378:metabolism; D006868:Hydrolysis; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity*; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity*; D016716:PC12 Cells; D010447:Peptide Hydrolases / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity*; D010766:Phosphorylation; D025941:Protein Interaction Mapping / Q000379:methods; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010748:Phosphopeptides; D019950:Mitogen-Activated Protein Kinase 1; D010447:Peptide Hydrolases