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Genes to Cells (2007) 12, 797-810. doi:10.1111/j.1365-2443.2007.01091.x
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Physical and functional interaction between mortalin and Mps1 kinase

 Masayuki Kanai1, Zhiyong Ma2, Hideki Izumi1, Song-Hee Kim1, Christopher P. Mattison3, Mark Winey3 and Kenji Fukasawa1,*

1 Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA
2 Department of Animal Infectious Diseases, Shanghai Veterinary Research Institute, Shanghai 200232, China
3 Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Mortalin is a member of Hsp70 chaperoning protein family involved in various cellular functions. Through the search of the kinases that mortalin physically interact with, we identified Mps1 as such a kinase. Mps1 kinase has been implicated in the regulation of centrosome duplication and mitotic checkpoint response. Mortalin binds to Mps1, and is phosphorylated by Mps1 on Thr62 and Ser65. The phosphorylated mortalin then super-activates Mps1 in a feedback manner. Mortalin has been previously shown to localize to centrosomes, and to be involved in the regulation of centrosome duplication. We found that centrosomal localization of mortalin depends on the presence of Mps1. Moreover, Mps1-associated acceleration of centrosome duplication depends on the presence of mortalin and super-activation by the Thr62/Ser65 phosphorylated mortalin.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Mortalin (also known as mot-2, Grp75, PBP-74, mtHsp70) is a member of the hsp70 family proteins, found in cytosol as well as various organelles including mitochondria, endoplasmic reticulum and cytoplasmic vesicles (Kaul et al. 2002). Mortalin is expressed in all cell types and tissues, and has been shown to exert diverse activities, including molecular chaperoning, oncogenic transformation, lifespan extension and attenuation of differentiation (reviewed in Kaul et al. 2002). It has recently been shown that mortalin localizes to centrosomes (Ma et al. 2006a). The centrosome, a major microtubule organizing center of an animal cell, plays a critical role in the establishment of cytoplasmic microtubule networks during interphase and assembly of bipolar spindles during mitosis. The centrosome is composed of a pair of centrioles and surrounding electron dense protein aggregates referred to as the pericentriolar material (PCM). Like DNA, the centrosome duplicates once in each cell cycle, which normally starts at the time of S-phase entry and is completed in late G2-phase (reviewed in Hinchcliffe & Sluder 2002; Fukasawa 2005). The centrosome is a non-membranous organelle, which makes a number of proteins freely accessible to this organelle. Indeed, the protein composition of PCM is dynamic, involving many different proteins; some of which are permanent constituents of centrosomes, while some localize to centrosomes in a cell (centrosome) cycle-dependent manner. The latter includes many of the proteins that control centrosome duplication. The comparative analysis of centrosomal proteins from unduplicated and duplicated centrosomes revealed that mortalin preferentially binds to duplicated centrosomes. Further studies have shown that mortalin starts to localize to centrosomes at late G1 phase prior to duplication, and then remains at duplicated centrosomes until mitosis at which time it dissociates from centrosomes (Ma et al. 2006a).

Like other molecular chaperoning proteins, mortalin is expected to bind to and modulate the functions of target proteins, including kinases. On this premise, we searched for the kinase(s) that physically interact with mortalin, and found that mortalin physically associates with Mps1 kinase. Mps1 (Mps1p in yeast nomenclature) was originally identified in the yeast temperature sensitive mutant that shows the defect in the duplication of spindle pole body (SPB, yeast centrosomes; reviewed in Winey & Huneycutt 2002). Mps1p phosphorylates several SPB components, including Spc98p, Spc110p and Spc42p in vitro, and the phosphorylation of these proteins in vivo has been shown to depend on the kinase activity of Mps1p. Spc98p, Spc110p and Spc42p are all involved in the assembly and duplication of SPB. In addition, it has been shown that Mps1p requires the presence of Cdc37 for its kinase activity (Schutz et al. 1997). Cdc37 is a molecular chaperone whose function, often in concert with Hsp90, is essential for the activities of many kinases in yeast (Bandhakavi et al. 2003). The mammalian Mps1 have been shown to localize to centrosomes, and to be stabilized by phosphorylation mediated by CDK2/cyclin E, a key kinase complex for initiation of centrosome duplication (Fisk & Winey 2001; Fisk et al. 2003). Mps1 has been implicated in initiation of centrosome duplication as a downstream target of CDK2/cyclin E as well as mitotic checkpoint response (Liu et al. 2003; Fischer et al. 2004; Jones et al. 2005; Schmidt et al. 2005; Grimison et al. 2006; Leng et al. 2006; Palframan et al. 2006 Zhao & Chen 2006). In this study, we show that mortalin physically interacts with Mps1, and is phosphorylated by Mps1 on Thr62 and Ser65. Mortalin, when phosphorylated, super-activates Mps1 in a feedback manner. We further found that Mps1 depends on the presence of mortalin and being super-activated by phosphorylated mortalin to drive centrosome duplication.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Identification of Mps1 as a mortalin-interacting kinase

Mortalin has been shown to function as a positive regulator of initiation of centrosome duplication (Ma et al. 2006a). As mortalin is a molecular chaperone, it is considered to control centrosome duplication through physically interacting with other proteins, and modulating their functions or enzymatic activities. In search of such proteins, especially kinases, the cell extracts were prepared from U2OS cells treated with hydroxyurea (HU), a potent DNA polymerase inhibitor (reviewed in Timson 1975). We used the HU-treated cells, because HU induces the cell cycle arrest at G1/S phase, where centrosome duplication is permissive. The lysates were immunoprecipitated with either anti-mortalin antibody or control IgG. Mortalin was successfully immunoprecipitated with anti-mortalin antibody (Fig. 1Ab). Since the kinases normally possess auto-phosphorylation activities, the immunoprecipitates (mortalin and mortalin-interacting proteins) were subjected to in vitro kinase assay in the presence of [32P-{gamma}]ATP (Fig. 1Aa). There was a readily detectable phospho-labeled protein band (MW {cong} 95 kDa) in the anti-mortalin antibody immunoprecipitates. As mortalin localizes to centrosomes, we searched for the known centrosomally localized kinases with MW {cong} 95 kDa, and we found that Mps1 kinase was present in the anti-mortalin antibody immunoprecipitates (Fig. 1Ad), suggesting that Mps1 may physically interact with mortalin either directly or indirectly as the components of a multi-protein complex.


Figure 1
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  		Figure 1  Identification of Mps1 as a kinase that interacts with mortalin. (A) The immunoprecipitates from U2OS cells treated with HU for 24 h using either anti-mortalin antibody or control mouse IgG were subjected to in vitro kinase reaction in the presence of [32P-{gamma}]ATP (a). The autoradiograph of the reaction mixtures fractionated in SDS-PAGE revealed the auto-phosphorylated band of 95 kDa. The immunoprecipitates and the lysates used for the immunoprecipitation were immunoblotted with anti-mortalin antibody (b and c, respectively). The immunoprecipitates were also immunoblotted with anti-Mps1 antibody (d). (B) Physical interaction between mortalin and Mps1. The cell lysates were immunoprecipitated with either rabbit anti-Mps1 (lanes 2 and 5) or mouse anti-mortalin antibody (lanes 8 and 11). The lysates were also immunoprecipitated by control preimmune-rabbit IgG (lanes 1 and 4) or preimmune-mouse IgG (lanes 7 and 10). The immunoprecipitates were then immunoblotted with either anti-Mps1 (lanes 4, 5, 7 and 8) or anti-mortalin antibody (lanes 1, 2, 10 and 11). Lanes 3, 6, 9 and 12, 10% input cell lysates used for immunoprecipitation. (C) Physical interaction between exogenously introduced mortalin and Mps1. FLAG-Mps1 and GFP-mortalin were co-transfected into 293T cells. FLAG-vector and GFP-vector were co-transfected as a control. The lysates prepared from the transfectants were immunoprecipitated with anti-GFP and anti-FLAG antibodies, and the immunoprecipitates were immunoblotted with anti-GFP antibody.

 
To corroborate the potential interaction between mortalin and Mps1, we conducted a co-immunoprecipitation assay (Fig. 1B). The cell lysates from U2OS cells were immunoprecipitated with rabbit anti-Mps1 antibody (lanes 2 and 5). As a control, IgG purified from pre-immune rabbit serum was used (lanes 1 and 4). The immunoprecipitates were then immunoblotted for mortalin and Mps1. Anti-Mps1 antibody immunoprecipitated mortalin (lane 2) as well as Mps1 (lane 5). The reciprocal experiment was performed using anti-mortalin antibody for immunoprecipitation (lanes 8 and 11). As a control, IgG purified from pre-immune mouse serum was used (lanes 7 and 10). The anti-mortalin antibody immunoprecipitated Mps1 (lane 8) as well as mortalin (lane 11). To exclude the possibility that the co-immunoprecipitation of mortalin and Mps1 is not due to the nonspecific activities of anti-mortalin and/or anti-Mps1 antibodies, we tested the interaction of ectopically expressed epitope-tagged mortalin and Mps1. Green fluorescent protein (GFP)-tagged mortalin and FLAG-tagged Mps1 were co-transfected into 293T cells, which are known for a high transfection efficiency. As a negative control, the GFP and FLAG vector plasmids were co-transfected. The lysates were prepared from the transfectants, and immunoprecipitated with anti-GFP and anti-FLAG antibodies (Fig. 1C). The immunoprecipitates were immunoblotted with anti-GFP antibody. Anti-FLAG antibody brought down GFP-mortalin in the lysates prepared from the cells co-transfected with FLAG-Mps1 and GFP-mortalin. Based on these observations, we concluded that mortalin and Mps1 physically interact in vivo.

Phosphorylation of mortalin by Mps1

The finding of physical interaction between Mps1 and mortalin prompted us to examine whether mortalin could serve as a substrate of Mps1. We performed in vitro kinase reaction with His6-tagged mortalin and either glutathione-S-transferase (GST)-Mps1 or GST-Mps1 mutant lacking the kinase activity (Mps1/KD) (Fisk et al. 2003; Fig. 2A). GST-Mps1 phosphorylated mortalin (lane 3), while GST-Mps1/KD failed to do so (lane 4). Thus, mortalin can serve as a substrate for Mps1 in vitro. The phosphoamino acid analysis of the in vitro phosphorylated mortalin showed that the phosphorylation occurred primarily on Ser and Thr residues (Fig. 2B). We next examined the Mps1-dependent phosphorylation of mortalin in vivo. GFP-tagged mortalin was co-transfected with either FLAG-Mps1 or FLAG-Mps1/KD into 293T cells. Similar levels of GFP-mortalin as well as FLAG-Mps1 and -Mps1/KD were expressed in the transfectants (Fig. 2C, top and 2nd panels). The lysates prepared from the transfectants were subjected to immunoprecipitation with anti-GFP antibody. The immunoprecipitates were then immunoblotted with anti-GFP (3rd panel), anti-phospho-Ser/Thr (4th panel) and anti-phospho-Tyr antibodies (bottom panel). Similar levels of GFP-mortalin were immunoprecipitated among the transfectants (lanes 2–4, 3rd panel). GFP-mortalin was strongly phosphorylated on Ser/Thr residues when FLAG-Mps1 was co-transfected (lane 3, 4th panel), while in the cells co-transfected with FLAG-Mps1/KD, GFP-mortalin was only minimally phosphorylated (lane 4, 4th panel) at the level similar to those co-transfected with the FLAG-vector (lane 2, 4th panel). Tyr phosphorylation on GFP-mortalin was not detectable by the method employed here. These findings show that Mps1 phosphorylates Ser and Thr residues in vitro as well as in vivo.


Figure 2
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  		Figure 2  Mps1 phosphorylates mortalin on Thr62 and Ser65. (A) His6-tagged mortalin were subjected to in vitro kinase reaction with either GST-Mps1 or GST-Mps1 kinase-dead mutant (GST-KD) in the presence of [32P-{gamma}]ATP. The autoradiograph is shown in the top panel. The bottom panel shows the Coomassie blue (CB)-stained gel. (B) His6-mortalin phosphorylated in vitro by GST-Mps1 in the presence of [32P-{gamma}]ATP were purified and subjected to a phosphor-amino acid analysis and visualized by autoradiography. Pi, free 32P; P-peptides, unhydrolyzed peptides; +, the sample loading origin. The positions of phospho-serine (P-Ser), phospho-threonine (P-Thr) and phospho-tyrosine (P-Tyr) standards visualized by ninhydrin staining were indicated. (C) Phosphorylation of mortalin by Mps1 in vivo. 293T cells were co-transfected with FLAG-vector + GFP-vector (lane 1), FLAG-vector + GFP-mortalin (lane 2), FLAG-Mps1 + GFP-mortalin (lane 3) and FLAG-Mps1/KD + GFP-mortalin (lane 4). The lysates prepared from the transfectants were immunoblotted with anti-GFP (top panel) and anti-FLAG (2nd panel) antibodies. The lysates were also subjected to immunoprecipitation with anti-GFP antibody. The immunoprecipitates were immunoblotted with anti-GFP (3rd panel), anti-phospho-serine/threonine (pS/T) (4th panel) and anti-phospho-tyrosine (pY) (bottom panel) antibodies. (D) His6-mortalin phosphorylated in vitro by GST-Mps1 was analyzed for phosphorylation sites by mass spectrometry. Thr62 and Ser65 were found to be phosphorylated. (E) His6-tagged wild-type mortalin (wt mortalin) and T62A/S65A alanine substitution mutant mortalin were subjected to in vitro kinase reaction with GST-Mps1 in the presence of [32P-{gamma}]ATP. The autoradiograph is shown in the top panel. The bottom panel shows the CB-stained gel. (F) 293T cells were co-transfected with FLAG-vector + GFP-mortalin (lane 1), FLAG-Mps1 + GFP-T62A/S65A unphosphorylatable mutant mortalin (lane 2) or FLAG-Mps1 + GFP-wt mortalin (lane 3). The lysates prepared from the transfectants were immunoblotted with anti-GFP antibody (top panel). The lysates were also subjected to immunoprecipitation with anti-GFP antibody and the immunoprecipitates were immunoblotted with anti-phospho-serine/threonine (pS/T) (2nd panel) and anti-GFP (bottom panel) antibodies.

 
We then attempted to identify the Ser and Thr residues phosphorylated by Mps1. To this end, unphosphorylated and in vitro phosphorylated His6-mortalin proteins were subjected to tryptic digestion and mass spectrometric analysis. The TurboSequest analysis of MS/MS spectra from the phosphorylated and unphosphorylated His6-mortalin identified a predicted mortalin tryptic peptide containing phospho-Thr62 and phospho-Ser65 (Fig. 2D). Thus, Mps1 phosphorylates mortalin on Thr62 and Ser65 in vitro. To confirm in vitro phosphorylation of Thr62 and Ser65 by Mps1, we performed in vitro Mps1 kinase reaction using the alanine-substitution mutant mortalin (T62A/S65A). As expected, T62A/S65A mutant mortalin failed to be phosphorylated by Mps1 in vitro (Fig. 2E, lane 1). To test whether this is also the case in vivo, GFP-tagged wild-type mortalin (wt mortalin) and unphosphorylatable alanine substitution mortalin mutant (mortalinT62A/S65A) were co-transfected with FLAG-Mps1 into 293T cells, and the lysates prepared from the transfectants were immunoprecipitated with anti-GFP antibody. The immunoblot analysis of the lysates showed that similar levels of FLAG-Mps1 were expressed in the transfectants (top panel, Fig. 2F). The anti-GFP antibody immunoprecipitates were immunoblotted with anti-pSer/Thr and anti-GFP antibodies. Similar levels of GFP-mortalin were immunoprecipitated (bottom panel, Fig. 2F). However, GFP-mortalinT62A/S65A failed to be detected by anti-pSer/Thr antibody (lane 2, 2nd panel), indicating that Mps1 phosphorylates mortalin on Thr62 and Ser65 in vivo.

Feedback super-activation of Mps1 by phosphorylated mortalin

We next examined the functional role of the mortalin phosphorylation mediated by Mps1. We first tested whether the phosphorylation of mortalin affects the physical interaction between Mps1and mortalin. To this end, GFP-wt mortalin, -T62A/S65A (unphosphorylatable mutant mortalin) and -T62D/S65D (phospho-mimetic mutant mortalin) were co-transfected with FLAG-Mps1 into 293T cells. As a control, the GFP-vector plasmid was transfected. Comparable levels of FLAG-Mps1 as well as GFP-mortalin were expressed (Fig. 3A, top and 2nd panels). The lysates prepared from the transfectants were subjected to immunoprecipitation using anti-GFP-antibody. Similar levels of GFP-mortalin were immunoprecipitated among the transfectants (bottom panel). Immunoblot analysis of the anti-FLAG antibody immunoprecipitates showed that similar levels of FLAG-Mps1 were co-immunoprecipitated among the transfectants (3rd panel), indicating that mortalin phosphorylation on Thr62 and Ser65 does not affect the physical interaction between mortalin and Mps1.


Figure 3
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  		Figure 3  Super-activation of Mps1 by mortalin. (A) The Mps1-binding affinity of mortalin is independent of Thr62 and Ser65 phosphorylation. 293T cells were co-transfected with FLAG-Mps1 + GFP-vector (lane 1), FLAG-Mps1 + GFP-wt mortalin (lane 2), FLAG-Mps1 + GFP-T62A/S65A mortalin (lane 3) and FLAG-Mps1 + GFP-T62D/S65D mortalin (lane 4). The lysates prepared from the transfectants were immunoblotted with anti-FLAG (top panel) and anti-GFP (2nd panel) antibodies. The lysates were also subjected to immunoprecipitation with anti-GFP antibody, and the immunoprecipitates were immunoblotted with anti-FLAG (3rd panel) and anti-GFP (bottom panel) antibodies. (B) Mortalin enhances the kinase activity of Mps1. 293T cells were co-transfected with FLAG-Mps1 + GFP-vector (lane 1), FLAG-Mps1 + GFP-wt mortalin (DNA 1 µg) (lane 2) and FLAG-Mps1 + GFP-wt mortalin (DNA 4 µg) (lane 3). The lysates prepared from the transfectants were immunoblotted with anti- FLAG (top panel) and anti-GFP (2nd panel) antibodies. The lysates were also subjected to immunoprecipitation with anti-FLAG antibody, and the immunoprecipitates were subjected to in vitro kinase reaction with [32P-{gamma}]ATP and myelin basic protein (MBP) as a substrate. The bottom panel shows the Coomassie blue (CB)-stained gel of the kinase reaction mixtures. (C) Super-activation of Mps1 by mortalin depends on Thr62/Ser65 phosphorylation. 293T cells were co-transfected with FLAG-Mps1 and GFP-vector (lane 1), FLAG-Mps1 + GFP-T62A/S65A mortalin (lane 2), FLAG-Mps1 + GFP-wt mortalin (lane 3) and FLAG-Mps1 + GFP-T62D/S65D mortalin (lane 4). The lysates prepared from the transfectants were immunoblotted with anti-FLAG (top panel) and anti-GFP (2nd panel) antibodies. The lysates were also subjected to immunoprecipitation with anti-FLAG antibody, and the immunoprecipitates were subjected to in vitro kinase reaction with [32P-{gamma}]ATP and MBP as a substrate. The bottom panel shows the CB-stained gel of the kinase reaction mixtures.

 
We next examined whether mortalin affects the kinase activity of Mps1. To test this possibility, we transiently co-transfected FLAG-Mps1 and different concentrations (1 and 4 µg) of GFP-mortalin into 293T cells. Immunoblot analysis showed that similar levels of FLAG-Mps1 were expressed in all the transfectants (top panel, Fig. 3B) and GFP-mortalin was expressed in a dose-dependent manner (2nd panel). The transfectants were subjected to immunoprecipitation using anti-FLAG antibody. The immunoprecipitated FLAG-Msp1 were then tested for the in vitro kinase activity with myelin basic protein (MBP) as a substrate (3rd panel). The kinase activity of Mps1 was increased in association with the increased level of GFP-mortalin, suggesting that mortalin enhances the kinase activity of Mps1. We next examined whether the phosphorylation status of mortalin influences its ability to super-activate Mps1. GFP-wt mortalin, -mortalinT62A/S65A and -mortalinT62D/T65D were co-transfected with FLAG-Mps1 into 293T cells. Comparable levels of FLAG-Mps1 (top panel, Fig. 3C) and GFP-mortalin (2nd panel) were expressed in the transfectants. The lysates prepared from the transfectants were immuno-precipitated with anti-FLAG antibody, and the immunoprecipitates were subjected to in vitro kinase assay. The T62A/S65A unphosphorylatable mutant mortalin almost completely lost the ability to super-activate FLAG-Mps1 (lane 2, 3rd panel), indicating that phosphorylation on Thr62 and/or Ser65 is critical event for mortalin to super-activate Mps1. Thus, Mps1 phosphorylates mortalin, which in turn super-activates Mps1 in a feedback manner.

Centrosomal localization of mortalin depends on the presence of Mps1

Mortalin has been shown to start to localize at centrosomes at mid-late G1 phase, and remain at centrosomes through the rest of the cell cycle (Ma et al. 2006a). Mps1, on the other hand, localizes to centrosomes throughout the cell cycle, although it is more abundantly found at centrosomes from mid-late G1 due to stabilization by CDK2/cyclin E (Fisk & Winey 2001). We next tested the mutual dependency of centrosomal localization of Mps1 and mortalin. We first examined whether the centrosomal localization of Mps1 depends on the presence of mortalin. To test this, we attempted to silence mortalin expression in U2OS cells by small interfering RNA (siRNA). We could silence the expression of mortalin to 10%–20% of the normal level (Fig. 4A). We then examined the centrosomal localization of Mps1 in the cells silenced for mortalin (mortalin-RNAi cells) and the control cells transfected with a randomized siRNA sequence. The mortalin-RNAi cells and control cells were transfected with either GFP-vector or GFP-Mps1, and co-immunostained with anti-GFP and anti-{gamma}-tubulin antibodies. The GFP-moiety was not found at centrosomes, as there was no co-localization of GFP and {gamma}-tubulin signals in the cells transfected with GFP-vector (data not shown). We found that there was no significant difference in centrosomal localization of GFP-Mps1 between the mortalin-RNAi and control cells (Fig. 4B). We also tested the centrosomal localization of endogenous Mps1 in the mortalin-RNAi and control cells (Fig. 4C). Similar to exogenously introduced GFP-Mps1, endogenous Mps1 localizes to centrosomes at similar efficiency in the mortalin-RNAi and control cells. Thus, centrosomal localization of Mps1 is independent of the presence of mortalin.


Figure 4
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  		Figure 4  Centrosomal localization of Mps1 is independent of mortalin. (A) siRNA-mediated silencing of mortalin expression. U2OS cells were transfected with either pSuper plasmid encoding siRNA specific for mortalin or pSuper plasmid with a randomized siRNA sequence (control) together with a plasmid encoding a puromycin-resistance gene at 20 : 1 molar ratio. After puromycin selection, the lysates were prepared from the transfectants and immunoblotted with anti-mortalin antibody. (B) Mortalin RNAi and control cells were transfected with GFP-Mps1, and co-immunostained with anti-GFP (green) and anti-{gamma}-tubulin (red) antibodies. The cells were also stained for DNA with DAPI (blue). Arrows indicate the positions of centrosomes. The insets show the magnified images of the areas indicated by arrows. Scale bar, 10 µm. (C) The mortalin RNAi and control cells were co-immunostained with anti-{gamma}-tubulin (red) and anti-Mps1 (green) antibodies. The cells were also stained with DAPI (blue). Arrows indicate the positions of centrosomes. The insets show the magnified images of the areas indicated by arrows. Scale bar, 10 µm.

 
We next tested whether centrosomal localization of mortalin depends on the presence of Mps1. We generated U2OS cells silenced for Mps1 expression by siRNA (Mps1-RNAi) (Fig. 5A). The Mps1-RNAi and control cells transfected with a randomized siRNA sequence were transfected with GFP-mortalin, and the transfectants were co-immunostained with anti-GFP and anti-{gamma}-tubulin antibodies (Fig. 5B). As previously shown (Ma et al. 2006a), GFP-mortalin was found at centrosomes in the control cells (panels a–c). In contrast, GFP-mortalin was no longer detected in the Mps1-RNAi cells (panels d–f). We also examined the centrosomal localization of endogenous mortalin in the Mps1-RNAi and control cells (Fig. 5C). Consistent with the GFP-mortalin study, endogenous mortalin was not detected at centrosomes in the Mps1-RNAi cells (panels e–h). Thus, the centrosomal localization of mortalin (or alternatively the stability of centrosomally localized mortalin) depends on the presence of Mps1.


Figure 5
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  		Figure 5  Centrosomal localization of mortalin depends on the presence of Mps1. (A) siRNA-mediated silencing of Mps1 expression. U2OS cells were transfected with either pSuper plasmid encoding siRNA specific for Mps1 or pSuper plasmid with a randomized siRNA sequence (control) together with a plasmid encoding a hygromycin-resistance gene at 20 : 1 molar ratio. After hygromycin selection for 2 weeks, the lysates were prepared from the transfectants and immunoblotted with anti-Mps1 antibody. (B) Mps1 RNAi and control cells were transfected with GFP-mortalin, and co-immunostained with anti-GFP (green) and anti-{gamma}-tubulin (red) antibodies. The cells were also stained with DAPI (blue). The magnified images of the indicated areas are shown on the bottom right. Scale bar, 20 µm. (C) Mps1 RNAi and control cells were co-immunostained with anti-{gamma}-tubulin (red) and anti-mortalin (green) antibodies. The cells were also stained with DAPI (blue). Arrows indicate the positions of centrosomes. The insets show the magnified images of the areas indicated by arrows. Scale bar, 10 µm.

 
We next tested whether Mps1-mediated phosphorylation on Thr62 and Ser65 is important for mortalin to localize to centrosomes. To this end, GFP-tagged wild-type, T62A/S65A and T62D/S65D mortalin were transfected into U2OS cells, and co-immunostained with anti-{gamma}-tubulin and anti-GFP antibodies. We found that there was no significant difference in the efficiency of centrosomal localization among wild-type, T62A/S65A and T62D/S65D mortalin (Fig. 6), indicating that the Thr62/Ser65 phosphorylation and/or phosphorylatability of mortalin by Mps1 does not play any role in the centrosomal localization of mortalin.


Figure 6
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  		Figure 6  Centrosomal localization of mortalin is independent of Thr62/Ser65 phosphorylation. U2OS cells were transfected with GFP-vector, GFP-wt mortalin, GFP-T62A/S65A or GFP-T62D/S65D. At 48 h post-transfection, cells were co-immunostained with anti-GFP (green) and anti-{gamma}-tubulin (red) antibodies. Cells were also stained with DAPI (blue). The arrows point to the positions of centrosomes. The insets show the magnified images of the areas indicated by arrows. Scale bar, 20 µm.

 
Mps1-dependent promotion of centrosome duplication by mortalin

It has been shown that Mps1 positively controls centrosome duplication (Fisk & Winey, 2001; Fisk et al. 2003). The finding that mortalin binds to and super-activates Mps1 in a feedback manner suggests that mortalin may act as a positive regulator of centrosome duplication via acting on Mps1. We first tested whether ectopic expression of GFP-wild-type (wt) mortalin and -mortalin phosphorylation mutants (T62A/S65A and T62D/S65D) affect centrosome duplication by the centrosome re-duplication assay. When cells were exposed to DNA synthesis inhibitor such as HU, cells undergo multiple rounds of centrosome duplication in the absence of DNA synthesis, resulting in generation of amplified (≥ 3) centrosomes (Balczon et al. 1995). However, this phenomenon preferentially occurs in cells lacking p53. In the presence of functional p53, p21 CDK inhibitor is up-regulated in a p53-dependent manner in response to the stress associated with exposure to DNA synthesis inhibitors (Tarapore et al. 2001), which in turn inhibits CDK2/cyclin E, a known initiator of centrosome duplication. U2OS cells retain wild-type p53, and thus centrosomes do not efficiently re-duplicate when exposed to HU. However, suppression of centrosome re-duplication by p53 can be alleviated by ectopic expression of the proteins that promote centrosome duplication, allowing centrosome re-duplication in the presence of HU. Thus, by exposing the U2OS cells to HU after transfection with mortalin, the positive effect of ectopically expressed mortalin on centrosome re-duplication can be assessed. However, because mortalin is known to participate in the regulation of the cell cycle (Kaul et al. 2002), ectopic expression of mortalin may affect the normal cell cycle progression, which may interfere with the accurate interpretation of data. To circumvent this problem, we used the modified centrosome re-duplication assay described previously (Fisk & Winey, 2001; Ma et al. 2006b) (Fig. 7A), in which U2OS cells were pre-treated with HU for 24 h. More than 95% of the cells could be arrested at G1/S transition and S-phase by the 24 h HU exposure (data not shown). We then transfected GFP-wt mortalin, -T62A/S65A, -T62D/S65D and a control GFP-vector into the pre-arrested U2OS cells in the presence of HU. After transfection, cells were cultured in the presence of HU for 40 h, and the number of centrosomes per cell in the GFP-positive cells was determined by co-immunostaining with anti-GFP and anti-{gamma}-tubulin antibodies (Fig. 7B, representative immunostaining images of the GFP-T62D/S65D-positive and negative cells in the same microscopic field are shown in Fig. 7C). In the control GFP vector-transfected cells, virtually no centrosome amplification was observed (~3%). In contrast, in the GFP-wt mortalin-transfected cells, there was a marked increase in the frequency of centrosome amplification (~28%) as shown previously (Ma et al. 2006a). Moreover, there was a further increase in the frequency of centrosome amplification in the GFP-T62D/S65D-transfected cells (~50%), while there was no induction of centrosome amplification in the GFP-T62A/S65A-transfected cells (~3%). Thus, over-expression of mortalin promotes centrosome re-duplication, and phosphorylation of Thr62 and Ser65 is critical for mortalin to promote centrosome re-duplication.


Figure 7
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  		Figure 7  Mutual dependency of mortalin and Mps1 to promote centrosome duplication. (A) Modified centrosome reduplication assay. U2OS cells were pre-arrested by HU exposure for 24 h. Cells are then transfected in the presence of HU, and the transfected cells are continued to be exposed to HU for 40 h and centrosome profiles are determined. (B) Thr62/Ser65 phosphorylation is critical for mortalin to drive centrosome re-duplication. U2OS cells pre-arrested with HU were transfected with GFP-vector, GFP-wt mortalin, GFP-T62A/S65A or GFP-T62D/S65D in the presence of HU. After transfection, cells were further incubated in the presence of HU for 40 h, and the centrosome profiles of the GFP-positive cells were determined by immunostaining {gamma}-tubulin. For each experimental sample, > 150 cells were examined. The results are shown as the average ± standard error from three experiments. (C) Representative immunostaining images of cells transfected with GFP-T62D/S65D after centrosome re-duplication assay. Cells were co-immunostained with anti-GFP (green) and anti-{gamma}-tubulin (red) antibodies. Cells were also stained with DAPI (blue). The areas of centrosomes are indicated (1–4). Compare the GFP-positive and -negative cells. The GFP-positive cells underwent centrosome re-duplication, resulting in centrosome amplification, while centrosome re-duplication was suppressed in the GFP-negative cells. The panels on the right show the magnified images of the area indicated. Scale bar, 20 µm. (D) Promotion of centrosome re-duplication by mortalin depends on the presence of Mps1. Mps1-RNAi and control cells pre-arrested with HU were transfected with GFP-vector, GFP-wt mortalin, GFP-T62A/S65A or GFP-T62D/S65D in the presence of HU. After transfection, cells were further incubated in the presence of HU for 40 h, and the centrosome profiles of the GFP-positive cells were determined by immunostaining {gamma}-tubulin. For each experimental sample, > 150 cells were examined. The results are shown as the average ± standard error from three experiments. (E) Efficient promotion of centrosome re-duplication by Mps1 depends on the presence of mortalin. Mortalin-RNAi and control cells pre-arrested with HU were transfected with either GFP-vector or GFP-Mps1 in the presence of HU. After transfection, cells were further incubated in the presence of HU for 40 h, and the centrosome profiles of the GFP-positive cells were determined by immunostaining {gamma}-tubulin. For each experimental sample, > 150 cells were examined. The results are shown as the average ± standard error from three experiments.

 
Because the phosphorylation of Thr62 and Ser65 is important for mortalin to drive centrosome duplication, we next tested whether the centrosome duplication regulatory activity of mortalin depends on the presence of Mps1. To this end, U2OS cells silenced for Mps1 expression (Mps1-RNAi cells) were subjected to the centrosome re-duplication assay described in Fig. 7A along with transfecting GFP-wt mortalin, -T62A/S65A, -T62D/S65D and a control GFP-vector (Fig. 7D). As a control, cells transfected with a randomized siRNA sequence were used. The control cells gave similar results with the experiment shown in Fig. 7B; both wt mortalin and T62D/S65D mutant promoted centrosome re-duplication. In contrast, the centrosome re-duplication promoting activities of both wt mortalin and T62D/S65D mutant were significantly compromised in the Mps1-RNAi cells, suggesting that mortalin depends on Mps1 for their activities to promote centrosome re-duplication.

Mps1 requires the presence of mortalin for its activity to control centrosome duplication

We next tested whether the presence of mortalin is necessary for Mps1 to drive centrosome duplication. To this end, U2OS cells silenced for mortalin expression (mortalin-RNAi cells) were subjected to the centrosome re-duplication assay along with transfecting either Mps1 or the control vector (Fig. 7E). As a control, cells transfected with a randomized siRNA sequence were used. In the control RNAi cells, exogenously introduced Mps1 accelerated centrosome re-duplication (~60%) as described previously (Fisk & Winey 2001; Fisk et al. 2003). However, in the mortalin-RNAi cells, the frequency of centrosome re-duplication was significantly reduced (~30%). Thus, the presence of mortalin is required for Mps1 to efficiently promote centrosome re-duplication.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We have previously shown that mortalin, a member of the heat shock chaperoning protein family, is involved in the regulation of centrosome duplication; ectopic expression of mortalin promotes centrosome re-duplication in the centrosome re-duplication assay (Ma et al. 2006a). Since it is common that a chaperoning protein forms a complex with kinases to modulate their activities, we searched for such kinases, and found that Mps1 physically interacts with mortalin. Mps1 has been shown to be one of the downstream targets of CDK2/Cyclin E for initiation of centrosome duplication. Mps1 is stabilized by CDK2/cyclin E, and believed to target key protein(s) for initiation of centrosome duplication (Fisk & Winey 2001). We further found that mortalin is phosphorylated by Mps1 on Thr62 and Ser65 in vitro and in vivo. Moreover, the centrosomal localization of mortalin heavily depends on the presence of Mps1; mortalin can be no longer detected at centrosomes in cells silenced for Mps1 expression. At present, we do not know whether Mps1 is required for mortalin to be recruited to centrosomes or to stably localize at centrosomes. Nevertheless, the Mps1-dependency of centrosomal localization of mortalin is consistent with the previous observations, in which Mps1 is stabilized by CDK2/cyclin E, and thus a higher level of Mps1 is found at centrosomes from mid-late G1 phase, at which time CDK2/cyclin E is activated, through the rest of the cell cycle, and degraded during mitosis by anaphase-promoting complex (Fisk & Winey, 2001; Liu et al. 2003; Palframan et al. 2006). Mortalin starts to localize at centrosomes in mid-late G1 prior to initiation of duplication, remains at duplicated centrosomes, and dissociates from centrosomes during mitosis (Ma et al. 2006a). Thus, centrosomal association/dissociation kinetics of mortalin mirrors that of Mps1. However, the phosphorylatability of mortalin by Mps1 does not appear to play any role in the physical interaction between mortalin and Mps1 or in the centrosomal localization of mortalin. For instance, the unphosphorylatable mortalin mutant can bind to Mps1 and localize to centrosomes as efficiently as wild-type and phospho-mimetic mutant mortalin.

We found that mortalin super-activates Mps1 through physical interaction. Interestingly, super-activation of Mps1 by mortalin does not take place simply by binding, but occurs when mortalin is phosphorylated on Thr62 and Ser65. For instance, the unphosphorylatable mortalin mutant, which can bind to Mps1 and localize to centrosomes in a similar efficiency with wt mortalin, fails to super-activate Mps1, while the phospho-mimetic mutant can efficiently super-activate Mps1. Thus, Mps1 binds to and phosphorylates mortalin, and in a feedback manner mortalin super-activates Mps1. It should be noted here that mortalin binding affects the kinase activity of Mps1, but not the stability of Mps1. For instance, we filed to detect any difference in the level of Mps1 between mortalin RNAi and control cells (data not shown). We further found that Mps1 depends on the presence of mortalin and being super-activated by mortalin to drive centrosome duplication. Ectopic expression of mortalin drives centrosome duplication in a manner-dependent on Mps1. However, the unphosphorylatable mutant can no longer exert such an activity. Moreover, Mps1 is unable to drive centrosome duplication in cells silence for mortalin expression.

We have previously shown that centrosomally localized p53 negatively controls centrosome duplication in the transactivation function independent manner (Shinmura et al. 2006). Moreover, mortalin drives centrosome duplication at least in part by promoting dissociation of centrosomally localized p53 (Ma et al. 2006a). In this study, we found that mortalin is also involved in the regulation of centrosome duplication via effecting the activity of Mps1. At this moment, we do not know whether mortalin's effects on centrosomal localization of p53 and Mps1 activity are related or separable for the regulation of centrosome duplication, which is currently under investigation in our laboratory.

In yeast, it has been shown that CDC37 molecular chaperoning protein super-activate Mps1 through the physical interaction, and Mps1 requires the physical interaction with CDC37 to drive SPB duplication (Schutz et al. 1997). The requirement of such a chaperoning protein for vertebrate Mps1 for centrosome duplication remains unknown. Our present finding raises an intriguing possibility that mortalin may act as a functional homolog of CDC37 for Mps1 to control centrosome duplication in mammalian cells. There has been a controversy for the involvement of Mps1 in the initiation of centrosome duplication; in a certain experimental system, Mps1 was shown not to be able to drive centrosome duplication (Stucke et al. 2002; Fischer et al. 2004). Our present finding, in which Mps1 requires the presence of mortalin to drive centrosome duplication, may provide a possible explanation for this discrepancy. The intracellular level of mortalin, like other chaperoning proteins, is critically influenced by the environmental stress and condition. Thus, the minor difference in the cell lines used in the experiments and experimental conditions and/or techniques can greatly influence the mortalin level, and thus the activity/ability of Mps1 for driving centrosome duplication.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cells, plasmids and transfection

293T and U2OS cells were maintained in the complete medium (DMEM supplemented with 10% fetal bovine serum, penicillin [100 units/mL], and streptomycin [100 µg/mL]) in an atmosphere containing 5% CO2. Transfection was performed using either FuGENE6 (Roche Applied Sciences) or Lipofectamine 2000 (Invitrogen) transfection reagent according to the manufacture's instruction. The plasmids with point mutations were generated through the PCR-based protocol. For construction of a plasmid encoding His6-tagged human mortalin, the PCR-amplified product was inserted in flame into a pRSET-A vector (Invitrogen) using BamHI-HindIII restriction sites. His6-tagged proteins were bacterially purified according to the protocol provided by the manufacturer (Invitrogen). Glutathione-S-transferase (GST)-tagged human Mps1 and Mps1 kinase dead (KD) fusion proteins (Fisk et al. 2003) were bacterially purified according to the manufacture's instructions (Amersham Pharmacia). For generation of a plasmid encoding GFP-mortalin, the BamHI-HindIII fragments of His6-mortalin were inserted in frame into a pEGFP-C1 vector (Clontech) using BglII and HindIII restriction sites. For construction of a plasmid encoding human Mps1 siRNA, the sequence corresponding to coding region 1439–1458 (5'-gacaccaagcagcaatacc-3') was inserted into pSUPER vector (OligoEngine). For construction of a plasmid encoding human mortalin siRNA, the sequence corresponding to coding region 30–48 (5'-ccgtctcgtgggcgccgca-3') was inserted into pSUPER vector.

Antibodies

The primary antibodies used in this study were anti-mortalin mouse monoclonal antibody (JG-1, Affinity Bioregents), anti-mortalin rabbit polyclonal antibody (H-155, Santa Cruz), anti-{gamma}-tubulin mouse monoclonal antibody (GTU-88, Sigma), anti-Mps1 rabbit polyclonal antibody (C-19, Santa Cruz), anti-GFP mouse monoclonal antibody (clone 7.1 plus 13.1, Roche), anti-FLAG mouse monoclonal antibody (M2, Sigma), anti-phospho-Ser/Thr antibody (Zymed) and anti-phospho-Tyr antibody (Zymed). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Santa Cruz), HRP-conjugated anti-rabbit IgG (Santa Cruz), Alexa 488-conjugated anti-mouse and anti-rabbit IgG, and Alexa 546-conjugated anti-mouse and rabbit IgG (Molecular Probes). Anti-{gamma}-tubulin rabbit polyclonal antibody was generated in our laboratory (Fukasawa et al. 1996).

Indirect immunofluorescence

Cells grown on coverslips were subjected to brief extraction (30 s) with extraction buffer (0.1% TritonX-100 in phosphate-buffered saline [PBS]), washed in PBS 3 times and fixed with 10% formalin/10% methanol for 20 min at room temperature. The cells were incubated with blocking solution (10% normal goat serum in PBS) for 1 h, then probed with primary antibodies for 1 h, and antibody–antigen complexes were detected with either Alexa 488- or Alexa 546-conjugated goat secondary antibody for 1 h at room temperature. The cells were washed 3 times with PBS after each incubation, and then counterstained with 4', 6'-diamidino-2-phenylindole (DAPI). Cells were examined under a fluorescence microscope (Nikon Microphot-FX) using a 60x objective lens. The fluorescence images were captured with SPOT CCD camera (Diagnostic Instruments Inc.).

Immunoblotting and immunoprecipitation

For immunoblotting, cells were lysed in lysis buffer (50 mM Tris–HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, phenylmethylsulfonyl fluoride [PMSF], 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 mM NaF and 100 µM Na3VO4). The lysates were boiled for 5 min with sample buffer (2% SDS, 10% glycerol, 60 mM Tris–HCl, pH 6.8, 5% ß-mercaptoethanol and 0.01% bromophenol blue), resolved by SDS-PAGE, and transferred onto Immobilon-P (Millipore Corp.) sheets. The blots were first incubated in blocking buffer (5% [w/v] non-fat dry milk in Tris-buffered saline [TBS] plus 0.05% Tween20) for 1 h. The blots were then incubated with primary antibodies for 16 h at 4 °C. After extensive wash, the blots were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The antibody–antigen complex was visualized by ECL chemiluminescence (Amersham Pharmacia). For immunoprecipitation, the lysates were pre-cleared by incubation with 20 µL of protein G- or A agarose beads for 1 h at 4 °C. The lysates were then incubated with antibody for 3 h at 4 °C. Protein G- or A agarose beads were then added to the lysates and the mixtures were further incubated for 2 h at 4 °C. The beads were pelleted and washed 3 times in wash buffer (0.1% NP-40, 50 mM Tris, pH 8.0, 150 mM NaCl, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 1 mM PMSF, 5 mM NaF, 100 µM Na3VO4). Antibody–antigen complexes bound to the beads were eluted in the sample buffer by boiling, and resolved by SDS-PAGE.

In vitro kinase assay

GST-Mps1 and His6-mortalin were mixed in the kinase buffer (50 mM Tris–HCl, pH7.5, 10 mM MgCl2, 0.5 mM DTT, 1 mM PMSF) including10 µM ATP, 10 µCi [32P-{gamma}]ATP and myelin basic protein (MBP) as a control substrate. For auto-phosphorylation assay of the immunoprecipitates of anti-mortalin antibody, the immunoprecipitates were mixed in the kinase buffer including 10  µM ATP, 10 µCi [32P-{gamma}]ATP and MBP. The kinase reaction was carried out for 30 min at 30 °C, and stopped by addition of sample buffer. The samples were then boiled for 5 min, resolved by SDS-PAGE and autoradiographed.

Phosphoamino acid analysis

His6-mortalin phosphorylated by GST-Mps1 in vitro in the presence of [32P-{gamma}]ATP was resolved by SDS-PAGE, eluted from the gel, and subjected to acid hydrolysis. The phosphorylated amino acids were separated by 2-D electrophoresis on a thin layer cellulose gel plate as described previously (Tokuyama et al. 2001).

Mass spectrometry

His6-mortalin phosphorylated by GST-Mps1 in vitro was resolved by SDS-PAGE. The His6-mortalin band on the gel identified by Coomassie blue staining was excised, and subjected to mass spectrometric analysis at the mass spectrometry core facility (University of Cincinnati).


   Acknowledgements
 
This study is supported by National Institute of Health (CA90522 and CA95925).


   Footnotes
 
Communicated by: Kozo Kaibuchi

* Correspondence: E-mail: kenji.fukasawa{at}uc.edu


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Accepted: 19 March 2007




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