Cellular Abundance of Mps1 and the Role of Its Carboxyl Terminal Tail in Substrate Recruitment

Abstract

Mps1 is a protein kinase that regulates normal mitotic progression and the spindle checkpoint in response to spindle damage. The levels of Mps1 are relatively low in cells during interphase but elevated in mitosis or upon activation of the spindle checkpoint, although the dynamic range of Mps1 expression and the Mps1 catalytic mechanism have not been carefully characterized. Our recent structural studies of the Mps1 kinase domain revealed that the carboxyl-terminal tail region of Mps1 is unstructured, raising the question of whether this region has any functional role in Mps1 catalysis. Here we first determined the cellular abundance of Mps1 during cell cycle progression and found that Mps1 levels vary between 60,000 per cell in early G 1 and 110,000 per cell during mitosis. We studied phosphorylation of a number of Mps1 substrates in vitro and in culture cells. Unexpectedly, we found that the unstructured carboxyl-terminal region of Mps1 plays an essential role in substrate recruitment. Kinetics studies using the purified recombinant wild type and mutant kinases indicate that the carboxyl-terminal tail is largely dispensable for autophosphorylation of Mps1 but critical for trans-phosphorylation of substrates in vitro and in cultured cells. Mps1 mutant without the unstructured tail region is defective in mediating spindle assembly checkpoint activation. Our results underscore the importance of the unstructured tail region of Mps1 in kinase activation.

The MPS1 Family of Protein Kinases

“…Nevertheless, Thr676 phosphorylation is required for Mps1 to function optimally in yeast and in human cells (33, 84, 85). Supporting the theory that autophosphorylation increases kinase activity, kinetic analysis of Mps1 phosphorylation revealed a lag phase in product formation that is eliminated by preincubation with cold ATP (80, 89). Therefore, Thr676 phosphorylation is likely a priming event for kinase activation.…”

“…Phosphorylation occurs predominantly at Ser/Thr sites, although Tyr phosphorylation is also observed in vitro (33, 79). Among a myriad of phosphorylation sites, Thr676 and Thr686 are observed to have significant effects on kinase activity (33, 79, 80, 84, 85, 89). Thr676 lies in the activation loop, whereas Thr686 is on the P+1 loop.…”

“…Another unexpected finding came from deletion analysis of the C-terminal region of MPS1, a 65-amino-acid region that is disordered in all known Mps1 structures. This region is susceptible to proteolysis, which may explain the disagreement in abundance measurements using N-terminal or C-terminal antibodies (89). Removal of the 65-amino acid (noncatalytic) tail reduces Mps1 transphosphorylation by about sixfold but has little impact on Mps1 autophosphorylation (89).…”

Targeting MPS1 Enhances Radiosensitization of Human Glioblastoma by Modulating DNA Repair Proteins

“…It has long been thought that mutation of the genes that control chromosome segregation during mitosis may explain the high rate of chromosomal instability and aneuploidy, which is characteristic of most solid tumors, including glioblastomas (GBMs) (4, 5). Monopolar spindle 1 ( MPS1 ) an essential SAC kinase, was originally identified as a protein kinase that is overexpressed in human tumor cells and a kinase associated with cell proliferation in mouse embryonic carcinoma cells (3, 4, 6, 7). MPS1 functions in several aspects of cell cycle control, including mitotic spindle assembly checkpoint activation, proper mitotic progression, centrosome duplication, chromosome alignment, error correction of kinetochore-microtubule attachment, and recruitment of SAC components to kinetochores (8–10).…”

TC Mps1 12, a novel Mps1 inhibitor, suppresses the growth of hepatocellular carcinoma cells via the accumulation of chromosomal instability

“…Mps1 phosphorylates serine, threonine and tyrosine in vitro; thus, it is a dual specificity protein kinase TTK (Lauze et al, 1995). Intermolecular autophosphorylation of Mps1 within its activation loop increases its kinase activity (Mattison et al, 2007;Sun et al, 2010). Mps1 is localized within centrosomes and kinetochores and associates with microtubules (Dou et al, 2003;Liu et al, 2003;Stucke et al, 2004).…”