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Originally published In Press as doi:10.1074/jbc.M707063200 on August 28, 2007

J. Biol. Chem., Vol. 282, Issue 42, 30553-30561, October 19, 2007
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Mps1 Activation Loop Autophosphorylation Enhances Kinase Activity*

Christopher P. Mattison{ddagger}, William M. Old§, Estelle Steiner{ddagger}1, Brenda J. Huneycutt{ddagger}2, Katheryn A. Resing§, Natalie G. Ahn§, and Mark Winey{ddagger}3

From the {ddagger}Molecular Cellular, and Developmental Biology, the §Department of Chemistry and Biochemistry, and the Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309

Received for publication, August 22, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 Thr675, Thr676, and Thr686, but phosphatase-treated and reactivated enzyme was only phosphorylated on Thr676. Mutation of Thr676 in hMps1 or the corresponding Thr591 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mono-polar spindle-1 (MPS1) gene was identified in Saccharomyces cerevisiae in a screen for mitotic spindle defective mutants (1) and was subsequently shown to encode an essential dual specificity, autophosphorylating protein kinase (2, 3). MPS1 is conserved (4, 5) and is required for a variety of functions during cell growth. The mitotic checkpoint function of Mps1 is conserved among several organisms including yeast, Xenopus laevis, Zebrafish, and humans (6–13). Recent evidence also suggests that human Mps1 (hMps1)4 is involved in a DNA damage checkpoint, functioning upstream of Chk2 (14). In S. cerevisiae, duplication of spindle pole bodies requires MPS1 at multiple steps (reviewed in Ref. 15). Similarly, centrosome duplication in mice and humans has been shown to require Mps1 (5, 16), but there is conflicting data on this point (9, 10). Roles for Mps1 in development and in the response to stress have been demonstrated in yeast, Drosophila, and Zebrafish (11, 17–21).

Highly controlled regulation of Mps1 kinase activity is essential for growth. For example, overexpression of MPS1 in S. cerevisiae results in inappropriate checkpoint activation (12, 22), whereas too little Mps1 activity is lethal (2). Mps1 is regulated at both the transcription level, in response to cell cycle progression and cell differentiation (3, 4, 11, 23), and by changes in protein stability (5, 24, 25). The activity of hMps1 rises to an extent greater than what can be explained by the increase in protein level alone during the G2/M transition (9, 23). Furthermore, checkpoint activation with nocodazole treatment of cells results in a ~30-fold increase in hMps1 activity, whereas the protein level remains similar to untreated mitotic cells (9). Increased hMps1 activity is correlated with more slowly electrophoretically migrating forms thought to be the result of phosphorylation (7, 9). These observations suggest that Mps1 is also regulated by changes in phosphorylation state.

Many kinases are activated when phosphorylated within the activation loop (reviewed in Refs. 26–28). In some cases, this can be catalyzed by autophosphorylation. For example, ERK8 autophosphorylates in vitro on both Thr and Tyr residues for activation, and this is also the likely method for activation in vivo (29). Although it is not clear which other kinases or regulatory subunits (if any) play a role in Mps1 activation, it is well known that Mps1 autophosphorylates in vitro (2, 4, 10, 23, 30–32). Tryptic digestion and sequencing of a radiolabeled catalytically active hMps1 fragment isolated from Escherichia coli indicates that Thr676 in the activation loop is a likely site for autophosphorylation (31).

In this study, we sought to identify and characterize the role of autophosphorylation in Mps1 activity. We find that both yeast and human Mps1 autophosphorylate in the activation loop at a conserved residue. Mutation of this residue (Thr676 in human and the corresponding Thr591 in yeast) decreases kinase activity in vitro and inhibits function in vivo. This observation clearly demonstrates that Mps1 autophosphorylation is essential for function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning, Mutagenesis, and Purification of hMps1—The pGEX-hMps1-ha plasmid and purification of GST-hMps1-ha protein have been described previously (16). The addition of GST did not adversely affect kinase activity, because proteolytic cleavage of this moiety from hMps1 did not significantly alter kinase activity. We determined that GST-hMps1-ha isolated from E. coli has specific activity of 11.6 pmol/min-mg toward myelin basic protein and 3.6 pmol/min-mg for autophosphorylation. This is comparable with the activity of the yeast enzyme isolated from yeast, which we estimate to be 34.9 pmol/min-mg for myelin basic protein and 3.11 pmol/min-mg for autophosphorylation.

hMPS1 mutations were generated by site-directed PCR mutagenesis using cloned Pfu DNA Polymerase (Stratagene). PCR fragments were digested with BspEI and SacI and ligated into the pGEX-hMps1-ha plasmid to generate mutant alleles. Combinatorial mutants were generated by sequential mutagenesis. For expression of hMps1 mutant alleles in cells, mutations were subcloned into the pHF36 plasmid (16) after digestion with BspEI and SacI. All of the mutant alleles were verified by sequencing, and the proteins were purified in a manner identical to wild type.

Cells and Culture Conditions—RPE1 and U2OS cells were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Hyclone) and penicillin-streptomycin (Invitrogen). Expression of wild type and mutant hMps1 alleles in vivo was done using the plasmid pHF36 (16). DNA was purified using MAXI preps (Qiagen) and transfected into cells using Effectene (Qiagen).

Kinase Assays—Unless otherwise noted, wild type and mutant kinase assays were performed with 200 ng of purified hMps1 in kinase assay buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 0.5 mM dithiothreitol, 1 mg/ml MBP, 0.1 mM ATP) for 20 min at 30 °C. For phosphatase treatment, hMps1 bound to Glutathione-Sepharose resin (Amersham Biosciences) was incubated in the presence of 4000 units of lambda phosphatase (New England Biolabs) at 30 °C for 2 h, followed by extensive washing in phosphate-buffered saline to remove the phosphatase and a final wash in 1x kinase assay buffer before assaying kinase activity. Coomassie-stained kinase bands were scanned using the Odyssey imaging system (Li-Cor) and normalized to autoradiography units to compensate for small errors in loading and more accurately reflect kinase activity.

Western Blotting—Human Mps1 protein was electrophoresed on 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride membrane. After blocking in TBST (0.1% Tween 20) with 6% milk and 1% bovine serum albumin, the blots were simultaneously probed with either 1:1000 rabbit polyclonal IgG sc-540 anti-hMps1 (TTK C-19; Santa Cruz) or 1:1000 anti-Mps1 NT clone 3–472-1 (Upstate%20Biotechnology">Upstate Biotechnology, Inc.), and mouse monoclonal IgG2b anti-Tyr(P) sc-7020 (p-Tyr PY99; Santa Cruz) or rabbit anti-Thr(P) (Zymed Laboratories Inc.). After three 5-ml washes with TBST, the blots were incubated with the appropriate secondary antibodies: Alexa 680-conjugated anti-rabbit or anti-mouse (Molecular Probes), IRDye800-conjugated anti-mouse, or anti-rabbit (Rockland Immunochemicals). Following three final 5-ml TBST washes, the blots were visualized using the Odyssey imaging system (Li-Cor).

Immunofluorescence Microscopy—Immunofluorescence microscopy was performed as described in Ref. 16 using the polyclonal anti-{gamma}-tubulin antibody (Sigma). The values for centrosome number reflect the averages ± standard deviation of three experiments in which at least 100 GFP-positive cells were counted for {gamma}-tubulin-positive spots.

Peptide Identification by Electrospray Ionization-Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS)–GST-hMps1-ha purified from E. coli was reduced with 10 mM dithiothreitol at 37 °C for 1 h, alkylated with 50 mM iodoacetamide at room temperature in the dark for 1 h, and then digested with 2% (w/w) sequencing grade trypsin (Promega), incubating overnight at 37 °C with gentle agitation. After acidification with formic acid, the samples were analyzed using a 4000 QTrap mass spectrometer (Applied Biosystems, Foster City, CA) interfaced with a NanoLC-2D HPLC (Eksigent, Dublin, CA), using a 75-µm x 15-cm Zorbax 300SB-C18 analytical nanocolumn (Agilent Technologies, Palo Alto, CA) for peptide separation. Buffer A was 0.1% formic acid and buffer B was 95% acetonitrile, 0.1% formic acid. An Endurance autosampler (Eksigent, Dublin, CA) was used to load the samples onto a 5-µm, 5 x 0.3-mm Zorbax 300SB-C18 trap column (Agilent Technologies) at 20 µl/min in 98% A, 2% B. After 5 min of washing, the trap column was switched in-line with the analytical column, and the gradient program and acquisition was started. Post-column auxillary flow was required for stable spray in negative mode, consisting of 80% 2-propanol at a flow rate of 300 nl/min from a Harvard syringe pump connected to the post-column flow with a Microtee tubing adapter (Upchurch, Oak Harbor, WA). The gradient program was run at a flow rate of 300 nl/min: 2% to 50% B in 82 min, 50–100% B in 15 min, and 100% B for 5 min. The column was recycled by washing in 98% A, 2% B.

Phosphopeptides were first detected by phosphopeptide precursor ion analysis, followed by a second targeted positive mode LC/MS/MS run to identify the peptides (33). In the first run, phosphopeptides were detected by precursor ion scanning in negative mode with an ion spray voltage of–2200 V applied to a PicoTip needle (FS360-75-15-N-20-C12; New Objective, Woburn, MA) monitoring for the marker ion PO–3 at–79 m/z, over a mass range of 500–1800 m/z, with Q1 set to low resolution and Q3 set to unit resolution. When the signal intensity of the precursor ion scan was above a threshold of 4000 cps, a high resolution scan in negative mode was performed for charge determination and accurate mass measurement. A mass list of candidate phosphopeptides was then generated and used as an inclusion list for targeted positive mode LC/MS/MS. This consisted of a linear ion trap survey scan, a high resolution scan, and MS/MS sequencing for targeted ions that matched masses on the inclusion list. MS/MS were searched with MASCOT (version 2.0, MatrixScience, London, UK) using a small data base of 20 standard proteins, including the sequence of the GST-hMps1-ha. Parent mass tolerance was 1.2 Da and MS/MS tolerance was 0.8 Da, with fixed modifications set to carbamidomethyl cysteine, variable methionine oxidation, and variable phosphorylation at Ser, Thr, and Tyr. MS/MS identifications with MASCOT scores above 25 were then manually validated for quality and phosphorylation site determination.

Matrix-assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) Analysis of Yeast Mps1—Yeast Mps1 was overexpressed with a plasmid borne GST fusion (described in Ref. 2) in wild type yeast cells from a galactose inducible promoter. GST-Mps1p was isolated from cell lysates by binding to glutathione beads (GE Healthcare). The samples were separated on an 8.5% SDS-PAGE gel and stained with imidazole/zinc to visualize GST-Mps1p, and the protein band was excised from the gel (34). Gel slices were destained with citric acid, reduced with dithiothreitol, and alkylated with iodoacetamide. Gel slices were then washed, dehydrated with acetonitrile, and completely dried by speed vac. The gel slices were reswelled in 15 µl trypsin/ammonium bicarbonate and allowed to digest overnight. Tryptic peptides were extracted from the gel slices, the supernatant was dried, and the sample was reconstituted in trifluoroacetic acid. The peptide sample was mixed with matrix and spotted onto a MALDI plate for analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Hyperphosphorylation of hMps1 Is Not Required for Activity–We isolated full-length recombinant hMps1 fused to both N-terminal GST and C-terminal hemagglutinin tags from E. coli to study the role of hMps1 autophosphorylation. Wild type GST-hMps1-ha contained multiple forms, some of which migrate more slowly in SDS-PAGE and are immunoreactive with anti-phosphotyrosine and anti-phosphothreonine antibodies (Fig. 1A). This suggested the kinase was highly phosphorylated and is consistent with changes in hMps1 electrophoretic migration previously noted in mitotic cells (7, 9, 10). In contrast, the kinase dead (hMps1-KD, D664A) enzyme isolated in the same manner showed only fast migrating forms of the kinase as seen previously (2, 3), strongly suggesting gel mobility retardation was due to autophosphorylation (Fig. 1A). To confirm that the decreased mobility of wild type hMps1 was due to phosphorylation, we treated isolated glutathione-Sepharose-bound hMps1 with lambda phosphatase. This treatment increased gel mobility and produced a banding pattern similar to that of the kinase dead protein (Fig. 1B). Phosphatase treatment also abolished immunoreactivity with anti-Tyr(P) and anti-Thr(P) antibodies. Thus, full-length hMps1 isolated from E. coli autophosphorylates. To determine whether autophosphorylation occurred in trans, we incubated kinase dead hMps1 with active hMps1. We found that hMps1-KD was phosphorylated, indicating that hMps1 autophosphorylation can occur through an intermolecular reaction (Fig. 1C). In our assay the kinase dead protein was phosphorylated with a stoichiometry of 0.8 mol phosphate/mol of hMps1, suggesting that a single residue was phosphorylated.

We compared the incorporation of 32P into MBP by hMps1 treated with phosphatase with that of mock treated hMps1 to investigate the effect of autophosphorylation on hMps1 kinase activity. Surprisingly, phosphatase-treated hMps1 had kinase activity comparable with the untreated hMps1 (Fig. 2, A and B). Further, for the duration of the kinase assay phosphatase-treated hMps1 remained in the faster migrating form and retained activity without recovery of anti-Thr(P) and anti-Tyr(P) immunoreactivity (Fig. 2C and data not shown). Conceivably, hMps1 autophosphorylation may not be required for activity and may serve other purposes in vivo. Alternatively, phosphorylated residues important for activity were missed by the phosphatase or were again phosphorylated by hMps1 after phosphatase treatment. If so, phosphorylation at these sites does not cause gel mobility retardation nor allow recognition by the anti-Tyr(P) or anti-Thr(P) antibodies we used. We sought to resolve this question by identifying hMps1 autophosphorylation sites and determining their importance for kinase activity.


Figure 1
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  		FIGURE 1.
Characterization of recombinant full-length hMps1 produced in E. coli. A, approximately 500 ng of either purified wild type (wt) or kinase dead (kd) GST-hMps1-ha isolated from E. coli was analyzed by SDS-PAGE followed by staining with Coomassie Brilliant Blue or transferred to polyvinylidene difluoride membrane and then immunoblotted with anti-hMps1, anti-Thr(P), and anti-Tyr(P) antibodies. Relative electrophoretic mobility of molecular mass markers is designated on the left of the Coomassie-stained gel. B, approximately 500 ng of bead-bound wild type hMps1 was treated with 4000 units of Lambda phosphatase ({lambda}) or mock treated and then analyzed as in A. C, purified wild type GSH bead-bound GST-hMps1-ha (60 ng) was incubated with hMps1-KD-ha (120 ng) in an in vitro kinase assay in the presence of [{gamma}-32P]ATP and visualized by SDS-PAGE and autoradiography.

 
hMps1 Autophosphorylates on Thr675, Thr676, and Thr686 within the Activation Loop—The kinase activity of phosphatase-treated hMps1 was comparable with untreated hMps1, indicating that much of the autophosphorylation was dispensable for activity, as has been suggested previously (10). Therefore, we mapped autophosphorylation sites on GST-hMps1-ha isolated directly from E. coli, after phosphatase treatment, and following reactivation by autophosphorylation. We used a method for phosphopeptide-specific precursor ion scanning, which is described in Ref. 33. Using trypsin-digested hMps1, we observed greater than 89% coverage of the protein analyzed directly after isolation from E. coli. Phosphorylation of residues in the activation loop between kinase subdomains VII and VIII are known to be important for activation of several kinases (26), and we focused our analysis on autophosphorylation sites detected within this region for the remainder of this study. Digestion of hMps1 with trypsin resulted in the generation of two peptides that spanned the activation loop, residues 662LIDFGIANQMQPDTTSVVK680 and 681DSQVGTVNYMPPEAIK696. Both of these peptides were detected by precursor ion scanning mass spectrometry, monitoring for the loss of PO–3 and subsequently sequenced for MS/MS identification and phosphorylation site mapping. The 662LIDFGIANQMQPDTTSVVK680 peptide was detected only in a phosphorylated form, whereas the phosphorylated form of the 681DSQVGTVNYMPPEAIK696 peptide was detected with variable stoichiometry of 5–60%. Both of these peptides were identified in an unphosphorylated form with a high degree of confidence in the hMps1-KD protein sample, but they were never detected in a phosphorylated form. This indicates that the phosphorylation sites we found in the wild type hMps1 protein were due solely to autophosphorylation. Three phosphorylated residues were found within these two peptides. Phosphorylation at residues Thr675, Thr676, and Thr686 (Fig. 3) was confirmed by the presence of fragment ions adjacent to and containing these phosphorylation sites in fragmentation spectra. Phosphorylation was observed at either Thr675 or Thr676 based on MS/MS of two MH2+2 ions corresponding to monophosphorylated peptide 662LIDFGIANQMQPDTTSVVK680, resolved by HPLC (Fig. 3, A and B). Doubly phosphorylated forms of this peptide were not observed in our analysis. MS/MS of the MH2+2 ion of monophosphorylated peptide 681DSQVGTVNYMPPEAIK696 revealed phosphorylation on Thr686 (Fig. 3C).


Figure 2
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  		FIGURE 2.
hMps1 retains kinase activity after dephosphorylation. A, approximately 100 ng of either phosphatase-treated or untreated purified GSH bead-bound GST-hMps1-ha was used for in vitro kinase assays in the presence of [{gamma}-32P]ATP. Bead-bound kinase was incubated in kinase assay buffer with 100 µM cold ATP and 1 mg of MBP at 30 °C for the indicated times. Following SDS-PAGE, phosphorylation of MBP substrate was analyzed by autoradiography (autorad). B, phosphoryl [{gamma}-32P]ATP transfer to MBP was normalized to hMps1 protein level and plotted against time for mock and phosphatase-treated GSH bead-bound GST-hMps1-ha. The graph shown is representative of at least three independent experiments. Phosphatase-treated hMps1 is represented by white squares, and untreated hMps1 is represented by black circles. Radiography units were normalized to the intensity of Coomassie-stained kinase bands for each time point using the Odyssey imaging system (Li-Cor, Lincoln, NE). Analysis of [{gamma}-32P]ATP incorporation by autophosphorylation generated similar results (not shown). C, approximately 100 ng of either phosphatase-treated or untreated purified GSH bead-bound GST-hMps1-ha was used for in vitro kinase assays lacking [{gamma}-32P]ATP for the indicated times as in A. Following SDS-PAGE, the samples were immunoblotted with anti-hMps1 and anti-phosphothreonine antibodies.

 


Figure 3
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  		FIGURE 3.
hMps1 autophosphorylates at Thr675, Thr676, and Thr686 in the activation loop. Each panel shows the MS/MS spectrum of two hMPS1 phosphopeptides, along with a summary of the observed fragment ions displayed on the peptide sequence. In each case, sufficient fragmentation was observed in proximity to the phosphorylated residue to unequivocally identify the site. The deduced phosphorylation sites are indicated with underlining. The b and y ions are indicated with 249 and 235, above or below the sequence respectively (an internal dot and a dot preceding the ion label indicates the presence of phosphate on the fragment ion). The symbol {Delta} indicates that the observed ion has a mass consistent with neutral loss of H3PO4 (98 Da), requiring that no form of the fragment ion with a mass corresponding to the unmodified serine or threonine was observed. A z indicatesayion missing the N-terminal NH3, which is sometimes observed when there is a Gln at the N terminus. A, identification of phosphorylation at Thr676 on the hMPS1 peptide 662LIDFGIANQMQPDTTSVVK680. The MS/MS was collected on a doubly charged parent (observed m/z = 1079.06 Da, theoretical m/z = 1079.01 Da) and identified by Mascot (Mowse 74; expectation score 3e-7). Because the adjacent amino acids can be phosphorylated, the critical ions for identifying the phosphorylation site are the unmodified y4 ion, the phosphorylated y5, and the {Delta}y5. If the peptide was heterogeneously phosphorylated, the unmodified y5 should have been seen, but it was not observed (the asterisk indicates an ion at 528.5 Da, which is too small to be the unmodified y5 ion). B, identification of phosphorylation at Thr675 on the hMPS1 peptide 662LIDFGIANQMQPDTTSVVK680. The MS/MS was collected on a doubly charged parent (observed m/z = 1079.61 Da, theoretical m/z = 1079.01 Da) and identified by Mascot (Mowse 48; exp. 1.1e-4). The critical ions for identifying the phosphorylation site are the {Delta}y6 and the unmodified y5 ion at 533.6, with no evidence for {Delta}y5. The phosphorylated y5 (predicted 613.3 Da) was not observed. Together with the data in A, this indicates that the two phosphorylated forms of this peptide are separating on the reverse phase chromatography. C, identification of Thr686 phosphorylation on 681DSQVGTVNYMPPEAIK696. The MS/MS was collected on a doubly charged parent (observed m/z = 915.03 Da, theoretical m/z = 914.91 Da) and identified by Mascot (Mowse 51, exp. 6.1e-05). The critical ions for identifying the phosphorylation site are the unmodified y ion series from y6 to y10 and the unmodified b ions b3 and b4, combined with the presence of the {Delta}y ion series, y11 to y13 and the {Delta}b ion series b6 to b10.

 
Phosphorylation sites were also mapped on phosphatase-treated enzyme that had been allowed to reactivate in the presence or absence of ATP. Based upon our previous results, autophosphorylation sites detected in the reactivated enzyme do not cause a significant mobility shift but are likely to reflect autophosphorylation sites required for activity. In the sample lacking ATP we could not detect phosphorylation sites in either of the activation loop peptides after phosphatase treatment. Phosphorylation on the 662LIDFGIANQMQPDTTSVVK680 peptide was detected after reactivation in the presence of ATP, and Thr676 was the major site of phosphorylation. The unphosphorylated form of this peptide was undetectable, indicating that it was again autophosphorylated with 100% stoichiometry. We were unable to detect phosphorylation in the 681DSQVGTVNYMPPEAIK696 peptide in the reactivated enzyme. The absence of phosphorylation on this peptide may be attributed to low peptide abundance and/or low stoichiometry of phosphorylation, making it difficult to detect reliably. Alternatively, phosphorylation at this site may require in vivo conditions or may be tied to de novo protein synthesis. In any case, of the six phosphorylatable residues within the hMps1 activation loop (see Fig. 6), we find that three Thr675, Thr676, and Thr686 can be modified by autophosphorylation. However, Thr686 on the 681DSQVGTVNYMPPEAIK696 peptide was only detected on de novo synthesized protein isolated from E. coli, at variable stoichiometry and not on reactivated enzyme. This suggests that phosphorylation of this residue is unlikely to be important for activity. In contrast, phosphorylation of the 662LIDFGIANQMQPDTTSVVK680 peptide was consistently detected at high stoichiometry in both the de novo and reactivated enzyme. Two residues, Thr675 and Thr676 were modified by phosphorylation on this peptide, but Thr676 was consistently the major site detected.

Mutation of Thr676 or Thr686 Reduces hMps1 Kinase Activity—Our mapping data suggested that Thr676 was an important site of autophosphorylation required for kinase activity, but it is possible that phosphorylation of Thr675 also plays a role. To test this we mutated hMps1 activation loop residues to determine the affect this had on kinase activity. We made changes at each of the phosphorylated activation loop residues Thr675, Thr676, and Thr686. Although we did not detect phosphorylation at Ser677, Ser682, or Tyr689, we also made changes at these sites within the activation loop as controls for comparison.

Mutation of the T676A residue reduced kinase activity, causing a 7-fold reduction in MBP phosphorylation rate compared with wild type (Fig. 4). The T676A mutation did not significantly decrease the autophosphorylation rate, causing only a 1.4-fold reduction. Interestingly, mutation of Thr675 resulted in a slight increase in both autophosphorylation and MBP phosphorylation. The control mutations S677A, S682A, and Y689F all resulted in weak effects in kinase activity (Fig. 4). Surprisingly, even though the apparent lack of phosphorylation did not affect kinase activity, mutation of the Thr686 residue greatly reduced kinase activity causing a 40-fold reduction in the MBP phosphorylation rate and a 4-fold reduction in the autophosphorylation rate (Fig. 4). The corresponding threonine residue is highly conserved in many kinases (26, 35), and the crystal structure of cAPK complexed with substrate suggests that this residue helps to orient residues important for substrate and ATP positioning in the active site (36). Therefore, we suspect that the decrease in activity caused by the T686A mutation is due to its role in catalysis rather than a lack of phosphorylation at this site. Our phosphorylation site mapping and mutation analysis of hMps1 indicates that phosphorylation of Thr676 within the hMps1 activation loop is important for full kinase activity.


Figure 4
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  		FIGURE 4.
Mutation of either Thr676 or Thr686 reduces hMps1 kinase activity. A, wild type (wt) and the indicated mutant hMps1 proteins (100 ng) were analyzed by in vitro kinase assay and visualized by SDS-PAGE and autoradiography. B, phosphoryl transfer of [{gamma}-32P]ATP to MBP or hMps1 autophosphorylation was normalized to wild type hMps1 protein level and activity and then graphed as the average plus standard deviation of error for three independent experiments. kd, kinase dead.

 
hMps1 Thr676 Autophosphorylation Is Important for Centrosome Duplication—To test the importance of phosphorylation at Thr676 in vivo, we determined what effect mutation of this residue has on centrosome duplication. We also tested the T675A and T686A mutations. A previous study has established that overexpression of the kinase inactive hMps1-D664A protein can prevent centrosome duplication (16). RPE1 cells were transfected with either wild type or mutant alleles of hMps1 fused to GFP and arrested in the S phase by the addition of hydroxyurea for 24 h. The number of centrosomes was then determined by immunofluorescence with {gamma}-tubulin antibodies as described in Ref. 16. Overexpression of either the control kinase inactive (D664A) or the T686A hMps1 mutant proteins resulted in the inhibition of centrosome duplication in roughly 50% of RPE1 cells, a 10-fold effect compared with cells expressing wild type hMps1, T675A, or GFP alone in which ~5% of cells had not duplicated their centrosomes (Fig. 5A). A milder but significant 4-fold effect was observed in cells expressing T676A, in which 19% of cells had unduplicated centrosomes after 24 h of S phase arrest (Fig. 5A).


Figure 5
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  		FIGURE 5.
Overexpression of T676A or T686A hMps1 mutant proteins inhibits centrosome duplication. A, RPE1 cells expressing GFP fusions to various hMps1 proteins were arrested in the S phase after the addition of hydroxyurea for 48 h. The cells were then fixed and analyzed by {gamma}-tubulin immunofluorescence and GFP epifluorescence. The centrosome (cen) number was determined by counting {gamma}-tubulin positive spots in GFP-positive cells expressing the indicated hMps1 proteins. Plotted in the graph are the results of at least three independent experiments after counting at least 100 GFP-positive cells. The averages and errors (standard deviations) are plotted in each case. B, centrosome number for U2OS cells expressing the various hMps1 proteins was analyzed as in A.

 
We sought to confirm these findings by testing the mutations in a second cell line using the same experimental conditions. Centrosome duplication in the U2OS cell line has been shown to be more resistant to expression of the hMps1-D664 protein for unknown reasons (16). Accordingly, we found that only 20% of U2OS cells overexpressing the D664A or T686A hMps1 proteins did not duplicate their centrosomes, whereas only 5% of cells expressing wild type hMps1 or T675A had not completed centrosome duplication (Fig. 5B). These effects are in agreement with our RPE1 cell results and with previously published data (16). The effect of U2OS cells overexpressing the T676A protein was also reduced. We could find no significant difference between cells expressing T676A or GFP alone, because 9 and 8% of cells respectively had unduplicated centrosomes (Fig. 5B).

Another assay for hMps1 activity relies on the observation that U2OS cells are known to undergo inappropriate centrosome reduplication when arrested in the S phase (9). Similar to previous results (16), we find that overexpression of hMps1 increases the reduplication rate in roughly 17% of cells, a 2-fold effect compared with GFP alone (Fig. 5B). The T675A mutation, which slightly increases activity in vitro, behaved like wild type and caused a 17% increase in reduplication. In contrast, only 9% of cells expressing hMps1-T676A or GFP alone had greater than two centrosomes. These results correlate with our in vitro kinase activity data and indicate that the less active T676A mutant protein cannot drive centrosome reduplication like wild type. Consistent with previous results, T686A overexpression effectively prevented reduplication in all but 2% of cells (Fig. 5B). This 8-fold effect compared with wild type is similar to cells expressing the kinase inactive D664A allele in which only 3% of cells had reduplicated centrosomes as previously reported (16).

Yeast Mps1 Residues Thr591 and Thr602 Are Required for Function in Vivo—Our analysis of hMps1 mutant proteins shows that Thr676 is an autophosphorylation site important for activity. We analyzed phosphorylation sites in the yeast Mps1 enzyme to determine whether phosphorylation of Mps1 activation loop residues is conserved. For this analysis, an overexpressed GST-Mps1 fusion protein (described in Ref. 2) was isolated from mitotically arrested wild type S. cerevisiae. Consistent with previously published data (2), the GST-Mps1 protein isolated in this manner migrates slowly compared with a kinase dead allele, suggesting that it is highly phosphorylated. MALDI-TOF and electrospray ionization-LC/MS/MS analysis of trypsin-digested Mps1 resulted in 75% sequence coverage and identified the activation loop peptide, 578IIDFGIANAVPEHTVNIYR596, in +80-kDa forms, indicating phosphorylation at one of two potentially phosphorylatable residues, Thr591 and Tyr595 (data not shown). These data confirm that both yeast and human Mps1 enzymes are phosphorylated within the same region of the activation loop. A second much larger activation loop containing peptide, 597ETQIGTPNYMAPEALVAMNYTQNSENQHEGNK628, was not detected in our analysis.


Figure 6
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  		FIGURE 6.
Conservation among Mps1 activation loop residues. Activation loops from the indicated kinases were aligned using the ClustalW 1.8 alignment program at the Baylor College of Medicine Human Genome Sequencing Center Search Launcher (searchlauncher.bcm.tmc.edu/multi-align/multialign.html). The alignment indicates conservation among Mps1 proteins at positions Thr686 and Tyr689, whereas Ser682 is similar. Residues Thr676 and Ser677 are conserved within metazoan Mps1 proteins. The numbers note specific residues for hMps1 and yeast Mps1, and P indicates a phosphorylated residue. Identical residues are indicated by a black background with white lettering, conserved residues are indicated by gray background with black lettering, and similar residues are indicated by gray background with white lettering.

 
To determine whether phosphorylatable residues within the yeast Mps1 activation loop are important for activity, we tested whether the following mutants: T591A (Thr675 or Thr676 equivalent residue in hMps1), Y595F, T598A, and T602A (Thr686 equivalent residue in hMps1), could complement a strain deleted for the essential MPS1gene. Strains carrying the Y595F or T589A mutations were able to grow, but those harboring the T591A and T602A alleles could not (Table 1). These in vivo assays are consistent with our hMps1 findings and suggest that the T591A and T602A mutations inhibit Mps1 kinase activity. We measured the kinase activity of these two mutant proteins and found that the T591A mutant protein had 5% of wild type activity, and the T602A mutant protein had no detectable activity (Table 1). In addition, when compared with wild type, both mutant proteins migrated more quickly on SDS-PAGE, consistent with reduced autophosphorylation. Finally, we attempted to generate phospho-mimetic alleles of yeast Mps1 by making T591D, T591E, or T602D mutations. Expression of the T591D and T591E mutations could rescue a strain deleted for the essential MPS1gene and allowed growth, further supporting the conclusion that autophosphorylation of this residue is needed for activity. Conversely, the T602D mutation could not rescue the mps1{Delta} strain, indicating that this mutation severely reduced activity (Table 1).


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  		TABLE 1
S. cerevisiae Mps1 activation loop autophosphorylation is required for function

 
Overexpression of Mps1 in yeast is known to induce a spindle assembly checkpoint arrest (22). Although expression of the T591D or T591E alleles complemented the lethality of an mps1{Delta} strain, they could not induce a checkpoint response. This suggests that the T591D or T591E proteins were not constitutively activated kinases with increased activity. We also constructed the phospho-mimetic T676D and T686D mutations in hMps1, but these mutations resulted in less active kinases (data not shown). However, the T676D mutation caused only a 2-fold reduction in kinase activity compared with the 7-fold reduction caused by the T676A mutation.

Our results with yeast Mps1 activation loop mutations parallel our hMps1 findings and indicate that analogous residues in the human and yeast Mps1 kinases are required for activity. Autophosphorylation of human Thr676 and yeast Thr591 is essential for full kinase activity, whereas the hMps1 Thr686 and yeast Mps1 Thr602 residues likely play a structural role important for catalytic activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Protein kinases are regulated by several mechanisms such as transcriptional control or changes in protein stability in response to various signals. Kinase activity can be further modulated by a variety of tactics including the binding of catalytic or inhibitory subunits, the presence of small molecules, changes in localization, and phosphorylation by upstream kinases or via autophosphorylation. Although the Mps1 protein level is known to be regulated by changes in transcription and protein stability that occur during cell cycle progression (3–5, 11, 23–25), the control of Mps1 catalytic activity is not well understood. Although MAPK has been shown to be important for Mps1 kinetochore localization in Xenopus (37) and microtubule binding can increase kinase activity roughly 4-fold in vitro (10), there are no known regulatory subunits or upstream activating kinases for Mps1. Changes in Mps1 autophosphorylation are thought to be responsible for altering Mps1 activity in response to cell cycle progression and checkpoint activation.

We report here that autophosphorylation of a highly conserved activation loop residue in both human (Thr676) and yeast Mps1 (Thr591) is required for full kinase activity. This is similar to ERK8 kinase for which activation loop residue autophosphorylation is the likely mechanism of activation in vivo (29). However, the possibility remains that activating phosphorylation of Mps1 in vivo may be performed by other kinases. For example, cAMP-dependent protein kinase A can autophosphorylate at Thr197 within the activation loop in vitro, but the more likely route to activation in vivo requires phosphorylation by phosphoinositide-dependent kinase 1 (38).

We show that the 662LIDFGIANQMQPDTTSVVK680 peptide in hMps1 was phosphorylated with 100% stoichiometry, on both de novo and reactivated enzyme, and our data are in agreement with a previously published report indicating that a catalytic fragment of hMps1 is phosphorylated on Thr676 (31). Although we also detected phosphorylation at Thr675, Thr676 was the major site of phosphorylation on this peptide. Combined with our data indicating that a single site on hMps1-KD is phosphorylated by wild type (Fig. 1), this suggests Thr676 as the site of autophosphorylation required for increased hMps1 activity. In support of this, mutation of this residue to alanine in hMps1 decreased in vitro activity at least 7-fold and when overexpressed in RPE cells caused an inhibition of endogenous hMps1 centrosome duplication function. Similarly, mutation of the corresponding residue in yeast, T591A, also decreased activity and prevented function in vivo, whereas phospho-mimetic mutations, T591(D/E), rescued function. In hMps1 the adjacent residue, Thr675, was also phosphorylated at a lower level, but mutation of this residue did not detectably affect centrosome duplication. Further, the T675A mutation resulted in an increase in activity in vitro, suggesting that phosphorylation of this residue antagonizes kinase activity. There are several possible reasons for this, but one that we favor is that mutation of this residue leads to increased autophosphorylation at the activating Thr676 residue, resulting in higher enzyme activity. There may be competition for phosphorylation at the Thr675 and Thr676 sites. It seems likely that phosphorylation at one of these two sites precludes phosphorylation at the other based on our inability to detect the 662LIDFGIANQMQPDTTSVVK680 peptide in a dually phosphorylated form (Fig. 3) and the data suggesting hMps1-KD is phosphorylated at a single site (Fig. 1). Although phosphorylation at Thr676 is likely required for proper alignment of catalytic residues, it seems that phosphorylation at Thr675 does not cause the active site reorientation required for increased activity. However, activity from a double T675A/T676A hMps1 mutation is about 2-fold less than the T676A mutation alone (data not shown), suggesting that in the absence of Thr676 phosphorylation, Thr675 phosphorylation can slightly increase activity.

The T686A in hMps1 and T602A in yeast Mps1 mutations also decreased kinase activity. We identified Thr686 as a site of autophosphorylation in de novo synthesized hMps1 with variable stoichiometry. It is not uncommon to find that phosphorylation sites can have variable stoichiometry even for the same kinase substrate pair. For examples, Pho85-dependent Pho4 sites are found at variable stoichiometry (39), and variable phosphorylation sites detected in mouse synaptosomal preparations can be interpreted as affecting the range of kinase activation (40). However, the Thr686 site was not detected when hMps1 was allowed to reactivate following phosphatase treatment, suggesting that phosphorylation of this site is not required for activity. Although this may be due to low stoichiometry at this site, lack of detectable phosphorylation at the Thr686 residue did not seem to affect kinase activity. Thus, it seems more likely that mutation of Thr686 results in the perturbation of the catalytic domain. This threonine residue is highly conserved in many kinases (26, 35), and the corresponding Thr in the crystal structure of cAPK complexed with substrate is thought to be critical for proper orientation of a conserved Lys residue important for anchoring substrate and ATP in the catalytic loop (36). Further, there is no evidence for phosphorylation of the corresponding residue in several other kinases including MTK1/MEKK4, a kinase with similarity to the hMps1 catalytic domain, but its mutation severely affects activity (41–43). On the other hand, Thr686 may not serve the same function in hMps1, and without a crystal structure of active Mps1 bound to ATP and substrate to clarify this point, it remains a possibility that phosphorylation of this residue at some low level is important for kinase activity.

Our data showing that autophosphorylation of Thr676 in the hMps1 activation loop can account for a 7-fold difference in activity in vitro cannot fully explain the observed rise in hMps1 activity during the G2/M transition or after mitotic arrest of cells with nocodazole (9, 23). It has previously been noted that hMps1 isolated from insect cells has a higher specific activity (10). Production of hMps1 in E. coli may lack proper chaperonin-assisted protein maturation. In yeast, Mps1 requires the Cdc37 chaperonin for kinase activity (44), and it is likely that Cdc37 acts to govern hMps1 autophosphorylation by maintaining proper folding during post-translational maturation. Recently, a new connection between the Hsp70 chaperonin family member Mortalin and hMps1 demonstrates that Mortalin is required for increased hMps1 activity and centrosome function (45). The kinase domain of hMps1 has been shown to bind microtubules, and this binding can increase kinase activity in vitro (10). These observations indicate that in addition to Mps1 regulation by autophosphorylation, proper hMps1 localization and interaction with specific partners can affect kinase activity. These multiple forms of regulation may allow for the temporal and site-specific activation of hMps1 at discrete compartments within the cell cycle.

Comparison of Mps1 activation loops from several organisms shows that residues we have identified as important for kinase activity are also highly conserved. Not surprisingly, Thr686 is conserved in each of the Mps1 proteins we have surveyed, and Thr676 is conserved in several metazoan Mps1 proteins including mouse, Xenopus, Zebrafish, and Drosophila (Fig. 6). It should be noted that in this region there is only one threonine, Thr591, in the yeast enzyme (Fig. 6). Although our alignment indicates a one-residue shift in autophosphorylation site, Thr591 in the S. cerevisiae Mps1 versus Thr676 in hMps1, it seems likely that enzyme activation proceeds through the same mechanism. Another point to consider is that the proximity of these two phosphorylated residues in the human enzyme, Thr675 and Thr676, may serve to more finely tune activity. The opposing effect of Thr675 and Thr676 mutations on hMps1 enzyme activity suggests a possible mechanism for an autophosphorylation-mediated switch to modulate kinase activity.

Although there is a strong correlation between slower migrating hyperphosphorylated forms of Mps1 and kinase activity (2, 7, 9, 32), our data indicate that much of the autophosphorylation is dispensable for catalytic activity. We find that hMps1 retains activity after phosphatase treatment and subsequently autophosphorylates at Thr676 for activation. Regardless, the high level of phosphorylation observed in vivo may serve another function such as proper localization, substrate or interacting protein recognition, or possibly as a signal for protein stability. There is evidence in yeast that Mps1 kinase activity and phosphorylation state is important for protein stability (24, 25).

Analysis of the data presented here would be greatly aided with the crystal structure of active hMps1. This study has demonstrated that autophosphorylation of conserved activation loop residues within Mps1 is essential for kinase activity. It is likely that additional factors such as interaction with specific binding partners can further alter activity in vivo.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM51312 (to M. W.) and was aided by a Fellowship Award from the Colorado Tobacco Research Program (to C. P. M.) and Department of Defense Fellowship Award DAMD17-03-1-0404 (to C. P. M.). This work was also supported by National Institutes of Health Grant R01 CA87648 (to K. A. R.) and by National Institutes of Health Training Grant GM07135 (to B. J. H. and E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Dept. of Biology, Niagara University, New York, NY, 14109. Back

2 Present address: Finnegan, Henderson, Farabow, Garrett & Dunner, LLP, 901 New York Ave., NW, Washington, D.C., 20001. Back

3 To whom correspondence should be addressed. E-mail: mark.winey{at}colorado.edu.

4 The abbreviations used are: h, human; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; MBP, myelin basic protein; LC, liquid chromatography; MS/MS, tandem mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; ha, hemagglutinin; GFP, green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank Shelly Jones, Alex Stemm-Wolf, Chad Pearson, and Yvette Bren-Mattison for critical reading of this manuscript.



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