Mps1 activation loop autophosphorylation enhances kinase activity.
The Mps1 protein kinase is required for proper assembly of the mitotic spindle, checkpoint signaling, and several other aspects of cell growth and differentiation. Mps1 regulation is mediated by cell cycle-dependent changes in transcription and protein level. There is also a strong correlation between hyperphosphorylated mitotic forms of Mps1 and increased kinase activity. We investigated the role that autophosphorylation plays in regulating human Mps1 (hMps1) protein kinase activity. Here we report that hyperphosphorylated hMps1 forms are not the only active forms of the kinase. However, autophosphorylation of hMps1 within the activation loop is required for full activity in vitro. Mass spectrometry analysis of de novo synthesized enzyme in Escherichia coli identified autophosphorylation sites at residues Thr(675), Thr(676), and Thr(686), but phosphatase-treated and reactivated enzyme was only phosphorylated on Thr(676). Mutation of Thr(676) in hMps1 or the corresponding Thr(591) residue within yeast Mps1 reduces kinase activity in vitro. We find that overexpression of an hMps1-T676A mutation inhibits centrosome duplication in RPE1 cells. Likewise, yeast cells harboring mps1-T591A as the sole MPS1 allele are not viable. Our data strongly support the conclusion that site-specific Mps1 autophosphorylation within the activation loop is required for full activity in vitro and function in vivo.
Mattison
Christopher P
CP
Molecular Cellular, and Developmental Biology, University of Colorado, Colorado 80309, USA.
Old
William M
WM
Steiner
Estelle
E
Huneycutt
Brenda J
BJ
Resing
Katheryn A
KA
Ahn
Natalie G
NG
Winey
Mark
M
D000483:Alleles; D019943:Amino Acid Substitution; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics; D018385:Centrosome / Q000201:enzymology*; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D020125:Mutation, Missense; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000201:enzymology* / Q000235:genetics; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; D013912:Threonine; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; C075840:TTK protein, human