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Originally published In Press as doi:10.1074/jbc.M208035200 on September 6, 2002

J. Biol. Chem., Vol. 277, Issue 46, 44013-44020, November 15, 2002
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Inhibitor-2 Regulates Protein Phosphatase-1 Complexed with NimA-related Kinase to Induce Centrosome Separation*

Masumi EtoDagger, Elizabeth Elliott, Todd D. Prickett, and David L. Brautigan

From the Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908

Received for publication, August 7, 2002, and in revised form, August 30, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Centrosome separation is regulated by balance of in situ protein kinase/phosphatase activities during the cell cycle. The mammalian NimA-related kinase Nek2 forms a complex with the catalytic subunit of protein phosphatase-1 (PP1C). This complex is located at centrosomes and has been implicated in regulation of the cycle of duplication and separation. Inhibitor-2 (Inh2) is an inhibitor protein specific for PP1C, and its expression level fluctuates during the cell cycle. Here we report cellular regulation of the Nek2·PP1C complex by Inh2. PP1C-binding segments of Nek2 were isolated by yeast two-hybrid screening using Inh2 bait. Inh2 indirectly associates with Nek2 via PP1C, which binds to both proteins, forming a bridged heterotrimeric complex. Double Ala mutation of the PP1C-binding site (KVHF) in Nek2 eliminated both PP1C and Inh2 interactions in both a yeast conjugation assay and an in vitro binding assay. The kinase activity of Nek2·PP1C was enhanced 2-fold by addition of recombinant Inh2, with EC50 = 10 nM. Immunofluorescence showed concentration of endogenous Inh2 at centrosomes and in a region surrounding the centrosomes. Transient expression of wild-type Inh2 increased by 5-fold dispersed/split centrosomes in fibroblasts, mimicking the phenotype produced by overexpression of Nek2. Deletion of the Inh2 C-terminal domain yielded Inh2-(1-118), which failed to interact with or activate the Nek2·PP1C complex, suggesting that the C-terminal region of Inh2 is required for regulation of the Nek2·PP1C complex. Thus, Inh2 can enhance the kinase activity of the Nek2·PP1C complex via inhibition of phosphatase activity to initiate centrosome separation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Centrosomes are structures in the eucaryotic cell that function as centers for organizing microtubules (see review in Ref. 1). During the cell cycle, centrosomes are first duplicated coincident with S phase and then undergo separation at the onset of mitosis and become the spindle poles for chromosome segregation. Recent discoveries have shown that protein phosphorylation is involved in regulation of the centrosome cycle (2). Kinases such as cyclin-dependent kinase (CDK)1 and NimA-related kinase (Nek2) have been implicated in regulation of the centrosome cycle. In particular, Nek2 is the human relative of the NimA protein Ser/Thr kinase first described from Aspergillus and localized at centrosomes (3). Ectopic expression of Nek2 gives separation without spindle formation, consistent with a loss of centrosome cohesion (4).

In addition to kinases, the protein phosphatase-1 (PP1) alpha -isoform is localized at centrosomes (5). Indeed, the catalytic subunit of PP1 (PP1C) binds to a PP1-binding motif (-KVHF-) in the C-terminal non-catalytic region of Nek2 (6). Coexpression of PP1 antagonizes Nek2 function via dephosphorylation of Nek2 itself and its substrates, indicating that PP1 acts a negative regulator for centrosome separation (6, 7). On the other hand, phosphorylation of PP1C by Nek2 reduces the phosphatase activity of the complex (6). Thus, the kinase·phosphatase complex of Nek2·PP1C is expected to function as a bistable switch described by Ferrell (8), potentially creating a double-negative feedback circuit for regulation of centrosome dynamics.

Phosphatase inhibitor-2 (Inh2) was first described in 1976 as a heat-stable protein that inhibited protein phosphatase activity (9). The specificity of Inh2 was later used for functionally defining type 1 versus type 2 Ser/Thr phosphatases (10). Several lines of evidence suggest involvement of Inh2 in cell cycle regulation. The expression level of Inh2 fluctuates during the cell cycle and is enhanced at mitosis (11). Nuclear localization of Inh2 occurs in parallel to S phase entry, and Inh2 is retained in the cytoplasm of cells at high density (12, 13). Glc8, a yeast homolog of Inh2, compensates for mutation of Ipl1 kinase in Saccharomyces cerevisiae, which suffers severe missegregation of chromosomes during mitosis (14). Thus, Inh2 seems to function in cell cycle regulation; however, the physiological targets of Inh2 are unknown. The N-terminal IKGI sequence of Inh2 is essential for potent inhibition of monomeric PP1C (15). PP1C associated with regulatory subunits such as the myosin-targeting subunit MYPT1 is insensitive to Inh2, and PP1C is thought to bind either to a targeting subunit or to Inh2, but not to both (16-18). The docking sites on PP1C for the IKGI sequence of Inh2 and the (R/K)(V/I)X(F/W) motif of other PP1C regulatory subunits have been mapped adjacent to one another on the back side, opposite the active site, suggesting a competition between Inh2 and regulatory subunits (19, 20). Here we report the discovery of binding of Inh2 to the Nek2·PP1 complex and show that this association activates Nek2 kinase. Our results implicate Inh2 as a regulator of the Nek2·PP1C complex and centrosome separation in cells, creating new potential links for coordination of cell cycle signals.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Two-hybrid Screening-- Human Inh2 residues 1-197 2 were cloned into pGBT10 and used to transform yeast strain HF7c, which was sequentially transformed with a randomly primed and size-selected mouse day 9 embryo DNA library cloned into vector pVP16 (Stan Hollenberg, Fred Hutchinson Cancer Center, Seattle, WA). As previously described (21), positives were selected by growth on triple drop-out medium (leu-/trp-/his-) and confirmed by expression of beta -galactosidase from an alternate promoter.

Proteins, Antibodies, and Immunoblotting-- The DNAs isolated from a two-hybrid screen were cloned into the BamHI-EcoRI sites of pGEX4T to produce glutathione S-transferase (GST) fusion proteins in the Escherichia coli BL21 strain. The DNA for human Inh2 residues 1-197 (12) was cloned into the pET30a vector (Novagen) for expression as a fusion protein with an N-terminal hexahistidine plus 44-residue S tagTM (His6S) and into the XbaI site of the pRK7-Myc vector for expression in mammalian cells with an N-terminal Myc epitope tag (sequence EQKLISEEDL) (18). The DNA fragment of Inh2 encoding residues 1-118 or 14-197 was prepared by PCR methods and inserted into the pET30 or pRK7-Myc vector. Full-length Nek2 was prepared by reverse transcription-PCR using RNA prepared from HeLa cells and was subcloned into the BamHI-EcoRI sites of the pRK7-HA3 mammalian expression vector, which added an N-terminal triple-hemagglutinin (HA) epitope tag (sequence YPYDVPDYA). Proteins were detected by immunoblotting using anti-pan PP1C antibody (Transduction Laboratories), affinity-purified sheep anti-Inh2 antibody (13), anti-Myc monoclonal antibody 9E10, and anti-HA monoclonal antibody 12CA5 as previously described (18). Rabbit lung extracts were prepared by homogenizing tissue in 50 mM MOPS (pH 7.0), 0.1 M NaCl, 1 mM EGTA, 1% Nonidet P-40 (IGEPAL CA-630), 0.4 mM Pefabloc, 1 µg/ml leupeptin, 10 µg/ml lima bean trypsin inhibitor, and 0.2% 2-mercaptoethanol; and the supernatant recovered after centrifugation was used as a source of native PP1C in binding assays.

Cell Culture, Transfections, and Nek2 Assays-- African green monkey kidney cells (COS-7) and human retinal pigment epithelial cells (ARPE19) were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% newborn calf serum (Invitrogen) and in DMEM/nutrient mixture F-12 with 10% fetal bovine serum (Invitrogen), respectively, in a humidified incubator at 37 °C with 5% CO2. For binding and kinase assays with N-terminally triple HA-tagged Nek2 (referred to as HA3-Nek2), each 150-mm dish of COS-7 cells (~106 cells) was transfected with 8 µg of DNA in 16 µl of NovaFECTORTM (VennNova, LLC, Pompano Beach, FL) for 24 h and then lysed in 2 ml of buffer containing 0.1% IGEPAL CA-630. Aliquots of 0.25 ml were mixed with 5 µg of His6S-Inh2, or 1.0 ml was mixed with 8 µg of monoclonal antibody 12CA5; and bound proteins were recovered by binding to 10 µl of S-proteinTM-agarose (Novagen) or 10 µl of protein-A agarose (Sigma), respectively. Beads were recovered and washed by centrifugation. Nek2 assay used the immunoprecipitate in 20 µl of reaction mixture consisting of 25 mM MOPS (pH 7.2), 10 mM MgCl2, 1 mM dithiothreitol, and 0.4 mM Pefabloc plus 0.2 mg/ml myelin basic protein (MBP) (Sigma) and 0.1 mM [gamma -32P]ATP (1 µCi/nmol) as substrates. After incubation at room temperature for 30 min, samples were analyzed by SDS-PAGE and PhosphorImager (Amersham Biosciences) analysis using ImageQuant software.

Centrosome Separation Assay-- A 100-mm stock plate of COS-7 cells was split 1:8 onto fibronectin-coated 22 × 22-mm coverslips seated in 35-mm tissue culture dishes. Cultures were incubated at 37 °C for at least 6 h before transfection. Each 35-mm culture was transiently transfected with 1 µg of either empty green fluorescent protein vector plasmid or plasmid encoding HA3-Nek2 or N-terminally Myc-tagged Inh2 (referred to as Myc-Inh2) using 3 µl of FuGENE 6 transfection reagent (Roche Molecular Biochemicals) in serum-free DMEM following the manufacturer's instructions. Cultures were allowed to express protein at least overnight (12 h) before fixing as described to preserve microtubule structure. Transfected cells were stained with antibodies directed against either HA and Myc as well as endogenous gamma -tubulin. Untransfected cultures of COS-7 cells in 35-mm dishes were prepared, and the growth medium was replaced either with fresh 10% newborn calf serum-supplemented DMEM or with 10 µM Taxol or 20 µM nocodazole solutions prepared in 10% newborn calf serum-supplemented DMEM. Cultures were incubated at 37 °C for 30 min for Taxol treatment and for 60 min for nocodazole treatment before fixation and staining. Cells were stained with antibodies directed against endogenous Inh2, gamma -tubulin, and beta -tubulin. A stage micrometer was used to calibrate the measurement tool in Openlab software (at ×60, 48 pixels = 10 µm; Improvision, Coventry, United Kingdom). Intercentriolar distances in transfected cells were measured with the calibrated measuring tool directly from contrast-enhanced images of gamma -tubulin staining. Only those cells with two clearly distinguished foci of gamma -tubulin staining were included in the analyses.

Immunofluorescence Microscopy-- Cells were rinsed once with 1.2× PEM (1 M PIPES, 50 mM EGTA, and 20 mM MgCl2) at 37 °C, fixed with methanol at -20 °C for 3 min, rinsed twice again with 1.2× PEM, and permeabilized with 0.1% Triton X-100 in 1.2× PEM for 5 min at room temperature. Cells were rinsed three times with phosphate-buffered saline (PBS) and incubated in 3% bovine serum albumin in PBS blocking solution for 1 h at room temperature. Various primary monoclonal and polyclonal antibodies, including sheep anti-Inh2, anti-Myc (9E10), mouse anti-HA (F7; Santa Cruz Biotechnology), rabbit anti-gamma -tubulin (Sigma), and Cy3-conjugated mouse anti-beta -tubulin (Sigma) antibodies, were diluted in 3% bovine serum albumin-containing PBS and applied to the coverslips for at least 1 h at room temperature or overnight at 4 °C. Cells were rinsed three times with PBS for 5 min each before staining with the appropriate secondary antibodies, including rhodamine-conjugated goat anti-rabbit, Oregon Green 488-conjugated goat anti-mouse, and Alexa 488-conjugated goat anti-sheep antibodies (Molecular Probes, Inc., Eugene, OR), diluted in 3% bovine serum albumin-containing PBS and 1 µg/ml Hoechst 33342 nuclear stain for 1 h at room temperature. Coverslips were rinsed again three times with PBS as described above and mounted onto glass slides with 10 µl of Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA). Background staining was determined by preparing identical coverslips without primary antibody.

Images of fixed cells were captured using Openlab software with a Nikon Microphot-SA epifluorescence microscope equipped with a Nikon Plan Apo ×60/1.4 oil immersion objective; filter sets for fluorescein isothiocyanate, Texas Red, and 4,6-diamidino-2-phenylindole fluorophores; and a Hamamatsu Orca C4742-95 digital camera. Raw data images were converted to 8-bit tiff images in Openlab and further processes using Adobe Photoshop Version 5.5.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphatase Inhibitor-2 Interacts with PP1C and Nek2 in Yeast Two-hybrid and Conjugation Assays-- Human phosphatase Inh2 was used as bait in a yeast two-hybrid screen, and five distinct positives from 3 × 106 clones in a mouse embryo library were retrieved. Among the clones were fragments of the known proteins Nek2 and spinophilin/neurabin II. Fig. 1A shows a schematic of the Nek2 structure with an N-terminal kinase domain and a C-terminal domain (CTD). The fragments of Nek2 recovered in the screen are shown as bars, and we note they both lie within the CTD and contain the coiled-coil domain (residues 306-330) plus the PP1C-binding motif, KVHF (residues 383-386). Other epitope-tagged full-length and CTD proteins that were used in this study, both wild-type and Ala-substituted, are shown in Fig. 1A.


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  		Fig. 1.   Yeast conjugation assay for binding of Inh2 to Nek2. A, schematic of the sequences for Nek2. Bars depicts the full-length protein; the two bars underneath are the regions recovered by screening. The kinase domain, coiled-coil region, and PP1C-binding sequence (KVHF) are indicated as boxes. The two bars indicate the cDNA fragments used. The double Ala mutant at the PP1C-binding site is denoted as AA. B, yeast conjugation assays. The cDNA inserts indicated above and to the left were fused with the DNA-binding domain in the pGBT9 (TRP1) vector or with the activation domain in the pVP16 (LEU2) vector. W303-1B (MATalpha , leu/trp/his) and HF7c (MATa, leu/trp/his) of S. cerevisiae were transformed with the vectors, respectively. A pair of transformants was mated overnight on a yeast/peptone/dextrose plate. The colonies were transferred onto the selective medium of double (leu-/trp-) or triple (leu-/trp-/his-) drop-out plates. Formation of diploid was detected as cell growth on the double drop-out plate (right panel), and the interaction between two insert proteins indicates diploid growth on the triple drop-out plate (left panel). At least two independent transformants gave the same results.

A yeast conjugation assay was employed to assess interactions between various forms of Inh2, the Nek2 CTD, and PP1C (Fig. 1B). Transformation and mating of various yeast MATalpha and MATa strains were verified on a control double drop-out plate, on which all formed colonies (right panel). On triple drop-out medium, colonies could form only if the specific pair of fusion proteins interacted. Each row is a different protein fused to a DNA-binding domain, and each column is a protein fused to the VP16 transcription activation domain. The entire first row and first column (left panel) showed no growth, as expected for the negative control, which was the PP2A alpha 4 subunit and which did not dimerize or interact with any of the other proteins. In the second row, wild-type Inh2 interacted with the wild-type Nek2 CTD, as expected from the screen, and interacted also with PP1C. The Nek2 CTD (second column) interacted strongly with PP1C, consistent with a previous report (6). Thus, in this assay, the Nek2 CTD interacted with both Inh2 and PP1C. However, if Val383 and Phe386 were substituted with Ala in the VXF motif to give Nek2 CTD(AA), interactions with both PP1C and Inh2 were lost entirely (third column). Likewise, there was no interaction of Nek2 CTD(AA) with any of the other forms of Inh2. Eliminating PP1C association with the VXF motif also completely prevented Inh2 interaction with the Nek2 CTD. The results suggest that Inh2 associated with PP1C, which at the same time was bound to Nek2. We imagine that the yeast version of PP1C, called Glc7, bridged the interaction of Inh2 with the targets recovered in the two-hybrid screen.

Different domains of Inh2 were required for interactions with different partners. With PP1C (Fig. 1B, left panel, fifth column), wild-type Inh2 and Inh2-(1-118), lacking 40% of the protein, interacted equally well, whereas Inh2-(14-197), without the 10IKGI13 sequence for PP1C recognition at the N terminus, interacted weakly. On the other hand, with the Nek2 CTD (second column), wild-type Inh2 interacted strongly, and Inh2-(14-197) interacted weakly, but Inh2-(1-118) did not interact, showing that the C-terminal region of Inh2 is required for this association. We compared the Nek2 CTD with another PP1 regulatory subunit, the N-terminal domain of the muscle glycogen-targeting subunit GM. There was only weak interaction with PP1C and no interactions with any of the forms of Inh2 in this assay. Our conclusions are that Inh2 interaction with the Nek2 CTD requires binding of PP1C and the C-terminal domain of the Inh2. Thus, the Inh2-PP1C interaction is different with monomeric PP1C compared with PP1C bound to the Nek2 CTD or with PP1C bound to GM.

Binding Assays Show Nek2·PP1C·Inh2 Complexes-- We used recombinant GST fusion proteins in pull-down assays to show interactions of the Nek2 CTD with Inh2 and PP1C. The Nek2 CTD and double Ala-substituted Nek2 CTD(AA) were expressed in bacteria as GST fusion proteins attached to glutathione-agarose beads and mixed with a rabbit lung extract as a source of native PP1C and endogenous Inh2. Bound proteins were eluted and analyzed by immunoblotting (Fig. 2A, upper and middle panels). PP1C bound to GST-Nek2 CTD, but not to GST-Nek2 CTD(AA) (Fig. 2A, upper panel). Inh2 binding exactly followed PP1C binding (Fig. 2A, middle panel). Substitution of only 2 residues in a motif known to interact directly with PP1C completely eliminated binding of Inh2. Equivalent amounts of the GST fusion proteins and GST itself as a negative control were bound to the beads and stained with Coomassie Blue (Fig. 2A, lower panel). We interpret these results as further evidence for formation of a Nek2 CTD·PP1C·Inh2 trimeric complex, with PP1C acting as a bridge between Nek2 and Inh2.


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  		Fig. 2.   Pull-down assay for binding of Inh2 to recombinant Nek2. A, interaction of GST-Nek2 CTD with endogenous PP1C and Inh2. GST, GST-Nek2 CTD, and GST-Nek2 CTD(AA) bound to glutathione-agarose were incubated for 2 h with a rabbit lung extract at 4 °C. Following three washes, the samples were analyzed by immunoblotting using anti-pan PP1C (upper panel) and anti-Inh2 (middle panel) antibodies. The amount of ligand protein was detected by Coomassie Blue staining (lower panel). Identical results were obtained in three independent experiments. B, interaction of HA3-tagged full-length Nek2 with His6S-tagged recombinant Inh2. His6S-tagged recombinant Inh2 was bound to S-protein-agarose beads. HA3-tagged full-length wild-type Nek2 or the double alanine mutant (AA) was expressed in COS-7 cells, and 10-µl aliquots of the extracts of these cells (1% of the total) were subjected to immunoblotting using anti-HA antibody (Cell Extract; upper panel). The rest of extracts were incubated for 2 h at 4 °C with the Inh2 beads. Following three washes, the HA3-Nek2 bound on Inh2 beads was detected by anti-HA immunoblotting (Inh2-Bound; lower panel). These results were replicated in two independent experiments. C, interaction of GST-Nek2 IBF·PP1C with recombinant Inh2. GST-Nek2 IBF or GST-alpha 4 (5 µg each) bound to glutathione-agarose beads was incubated with 0.1 ml of 0.1 µM His6S-tagged purified recombinant Inh2 at room temperature for 5 min in a pull-down binding assay. Following three washes, the samples were analyzed by Western blotting with affinity-purified anti-Inh2 (upper panel) and anti-pan PP1C (lower panel) antibodies. The GST-Nek2 IBF·PP1C complex was prepared by a 2-h incubation with a rabbit lung extract and washing (right two lanes). Identical results were obtained in three independent experiments.

Association of Inh2 with full-length Nek2 was tested by transient transfection and affinity purification. Full-length Nek2 with an N-terminal triple-tandem hemagglutinin epitope tag was transiently expressed in COS-7 cells. The wild-type and Ala-substituted versions of Nek2 (Fig. 1A) were expressed at similar levels. Samples of these cell extracts and a control extract from untransfected cells were subjected to anti-HA immunoblotting to show the relative expression levels of HA3-Nek2 proteins (Fig. 2B, upper panel). Human recombinant Inh2 was produced in bacteria as a His6S fusion protein and purified to homogeneity. This His6S-Inh2 protein was bound to S-proteinTM-agarose and distributed into three parallel columns for affinity chromatography with the different cell extracts. The bound proteins from each column were eluted and analyzed by immunoblotting using anti-HA antibody (Fig. 2B, lower panel). The HA3-tagged wild-type Nek2 protein bound to Inh2, but Ala-substituted HA3-Nek2 did not. Only Nek2 with an intact VXF motif for interaction with PP1C could bind to Inh2.

Fig. 2C shows the indirect interaction of His6S-Inh2 with GST-Nek2 Inh2-binding fragment (IBF). His6S-Inh2 (0.1 µM) was mixed with GST-Nek2 IBF on glutathione-agarose beads. Bound His6S-Inh2 was analyzed by Western blotting. GST-alpha 4 was used as a negative control. His6S-Inh2 failed to associate with GST-Nek2 IBF, whereas the GST-Nek2 IBF·PP1C complex prepared by preincubation with a lung extract coprecipitated with His6S-Inh2 (Fig. 2C). The results of binding assays and the yeast conjugation assay indicate that Inh2 is capable of interaction with PP1C on Nek2.

Centrosomal Localization of Endogenous Inh2-- Nek2 is known to be localized at centrosomes, and the association of Inh2 with Nek2 predicts that Inh2 should appear at centrosomes in living cells. By immunofluorescence microscopy, we found that endogenous Inh2 was concentrated into a cloud in the perinuclear region in COS-7 cells that were fixed under conditions to preserve microtubules (Fig. 3A). Centrosomes were embedded within this cloud of Inh2 and were located by staining with anti-gamma -tubulin antibody (inset, yellow arrow). The cloud of Inh2 was centered at the microtubule-organizing center and depended on microtubule organization. Treatment of COS-7 cells briefly with nocodazole to disrupt microtubules dispersed the cloud of endogenous Inh2 and the fibrillar staining for beta -tubulin (Fig. 3B, panels c and d). Longer exposure to nocodazole made the Inh2 staining even more diffuse (data not shown). On the other hand, stabilizing microtubules by adding Taxol to the cells enlarged the zone of bright staining for Inh2; and upon closer inspection, the Inh2 staining appeared in part to overlap microtubules emanating from the centrosome (Fig. 3B, panels a and b). Association of Inh2 with centrosomes was more evident in ARPE19 cells that did not have a cloud of Inh2 (Fig. 3, C and D). Instead, staining of Inh2 was concentrated into discrete foci near the nucleus (Fig. 3C), which exactly colocalized with gamma -tubulin as a marker for the location of the centrosomes in double-stained cells (data not shown). These results show that endogenous Inh2 was localized at centrosomes and, in some cells, was concentrated into a cloud surrounding the centrosomes by a microtubule-dependent mechanism.


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  		Fig. 3.   Localization of endogenous Inh2 around microtubule-organizing centers in COS-7 and ARPE19 cells. COS-7 (A and B) and ARPE19 (C and D) cells were fixed and stained as described under "Experimental Procedures." Endogenous Inh2 is shown in green and DNA in blue. A, untreated COS-7 cells were stained with anti-Inh2 and anti-gamma -tubulin antibodies. The inset is centered on the position marked with an arrow (at the same magnification; bar = 5 µm), with the anti-gamma -tubulin stain shown in red to locate the pair of centrosomes. B, COS-7 cells were treated with Taxol (panels a and b) or nocodazole (panels c and d) prior to the fixing step. The specimen was stained with anti-Inh2 (panels a and c) and anti-beta -tubulin (panels b and d) antibodies. Bar = 20 µm. C and D, ARPE19 cells was stained with anti-Inh2 antibody. The area in the box in C is enlarged at D. The anti-gamma -tubulin stain was overlapped with the anti-Inh2 stain in the specimen subjected to the double immunostaining (data not shown). Bar = 5 µm.

Activation of Nek2 by Inh2-- We assayed the effects of added Inh2 on the kinase activity of a Nek2·PP1C complex. In this complex, PP1C suppressed kinase activity by dephosphorylation and inactivation of Nek2. The HA3-Nek2·PP1C complex was prepared by transient transfection and anti-HA immunoprecipitation from COS-7 cells. Nek2 kinase activity was assayed by incorporation of 32P from [gamma -32P]ATP into the exogenous substrate MBP. Addition of His6S-tagged recombinant Inh2 produced a dose-dependent activation, with EC50 ~ 10 nM (Fig. 4). The maximum activity given by addition of 1 µM His6S-Inh2 was the same as the activity induced by addition of 1 µM microcystin LR, a potent PP1/PP2A inhibitor (data not shown), suggesting the full activation of the HA3-Nek2·PP1C complex induced by Inh2. This effect is consistent with the high affinity binding of His6S-Inh2 to PP1C complexed to HA3-Nek2. We propose that inhibition of PP1C by Inh2 resulted in higher specific activity of Nek2. The anti-HA immunocomplex might be a mixture of HA3-Nek2·PP1C plus HA3-Nek2 unbound to PP1C; therefore, basal activity without His6S-Inh2 is due to free HA3-Nek2.


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  		Fig. 4.   Activation of Nek2 kinase by recombinant Inh2. Kinase assays were performed using anti-HA immunoprecipitates from COS-7 cells transiently transfected with a vector encoding HA3-Nek2. [gamma -32P] ATP (0.1 mM) plus MBP (0.2 mg/ml) were added as substrates in the presence of 10 mM Mg(OAc)2, 1 mM dithiothreitol, 0.4 mM Pefabloc, and 25 mM MOPS-NaOH (pH 7.0). The reaction was started by addition of ATP within 2 min after His6S-tagged recombinant Inh2 was added at the indicated concentrations. Kinase activity was measured as incorporated 32P radioactivity in MBP by PhosphorImagerTM analysis following SDS-PAGE. The 100% value indicates the kinase activity without Inh2, and normalized activities are shown (mean ± S.E., n = 4).

Structural Requirements for Inh2 Binding and Activation of Nek2·PP1C-- We tested for binding and activation of Nek2·PP1C complexes with 1) Inh2 (residues 1-198), essentially the full-length wild-type protein; 2) C-terminally truncated Inh2-(1-118), which lacks two sites for interaction with PP1C (17, 22); and 3) N-terminally truncated Inh2-(14-197), which lacks the IKGI motif (residues 10-13) required for binding to monomeric PP1C (15). These His6S-Inh2 proteins were produced in bacteria, purified to homogeneity, and immobilized on S-proteinTM-agarose for affinity chromatography. An extract of COS-7 cells expressing HA3-Nek2 was split and subjected to affinity chromatography using parallel columns of the different recombinant Inh2 proteins. As shown in Fig. 5A, all three forms of His6S-Inh2 bound both HA3-Nek2 and PP1C from extracts, but the relative recovery of these proteins was quite different. The His6S-tagged wild-type Inh2 protein bound a substantial amount of PP1C relative to its abundance in the extract. By comparison, the His6S-tagged wild-type Inh2 protein bound a much smaller fraction of total HA3-Nek2 from the extract. This hinted that of all the endogenous PP1C that could bind to immobilized Inh2, only a minor pool was associated with HA3-Nek2. Deletion of the IKGI sequence severely reduced binding of PP1C to His6S-Inh2-(14-197) to levels that were barely detectable, as expected. Likewise, only low levels of HA3-Nek2 bound to His6S-Inh2-(14-197), consistent with PP1C participating in the interaction. Compared with these results, we were surprised by the robust recovery of HA3-Nek2 by immobilized His6S-Inh2-(1-118), which yielded much more than even wild-type Inh2. The level of PP1C recovered was lower than with wild-type Inh2, making the ratio of HA3-Nek2 to PP1C very high. This suggests that His6S-Inh2-(1-118) preferentially bound to HA3-Nek2·PP1C complexes compared with either wild-type Inh2 or Inh2-(14-197).


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  		Fig. 5.   Mapping of the structural requirement of Inh2 for interaction with the Nek2·PP1C complex. A, pull-down assay for binding of the truncated Inh2 mutant to HA3-Nek2·PP1C. Different versions of tagged His6S-Inh2 (5 µg of each), wild-type (WT; residues 1-197), N-terminally truncated (residues 14-197), or C-terminally truncated (residues 1-118), were expressed in bacteria and bound to S-protein-agarose beads. HA3-tagged full-length Nek2 was expressed in COS-7 cells, and extracts of these cells were incubated for 2 h at 4 °C with the Inh2 beads, which were washed and subjected to immunoblotting. Samples of the COS-7 cell extract (as a reference for the amount of input protein) and S-protein beads without bound Inh2 (labeled blank as a negative control) were analyzed with the samples bound to different Inh2 proteins using monoclonal anti-HA (upper panel) and anti-pan PP1C (middle panel) antibodies. As an additional control, the different His6S-Inh2 proteins in the samples were stained with Coomassie Blue (lower panel). These results were replicated in four independent experiments. B, activation of Nek2 kinase by different forms of Inh2. Assay of Nek2 kinase isolated by immunoprecipitation from transiently transfected COS-7 cells was carried out as described in the legend to Fig. 4, with the inclusion of different versions of His6S-tagged purified recombinant Inh2 (100 nM): residues 1-198 (wild-type (WT)), residues 14-198, and residues 1-118. The relative kinase activities (mean ± S.E., n = 3) are compared.

We assayed activation of Nek2 kinase in HA3-Nek2·PP1C complexes recovered by immunoprecipitation in response to addition of the different His6S-Inh2 proteins used for affinity purification (Fig. 5B). Addition of 100 nM His6S-Inh2 in these assays gave a >2-fold increase in Nek2 kinase activity (n = 3; p < 0.001), measured by phosphorylation of MBP as substrate. The His6S-tagged N-terminally truncated Inh2-(14-198) protein, which showed low recovery of PP1C and HA3-Nek2 by affinity chromatography (Fig. 5A), nonetheless still produced a significant activation of HA3-Nek2 (p < 0.05). A paradoxical result was that His6S-Inh2-(1-118) had no significant effect on Nek2 activity, even though it yielded the most HA3-Nek2 by affinity chromatography. Thus, binding and activity did not correlate to one another. The results in Fig. 5 (A and B) together show that Inh2-(1-118) can bind to the Nek2·PP1C complex, but that the interaction does not produce activation of the kinase. We conclude that C-terminal residues 120-198 of Inh2 are required for inhibition of PP1C bound to Nek2.

Centrosome Separation and Inh2 Localization in Fibroblasts-- We demonstrated that Inh2 participates in regulation of Nek2 in living cells. Centrosome separation was assayed in COS-7 cells and in cells transiently transfected with either HA3-Nek2 or Myc-Inh2. Staining for gamma -tubulin visualized the location of centrosomes, seen as a closely spaced pair of intensely stained foci near the nucleus in control cells (Fig. 6, A, C, and E, yellow arrows). The distance between these brightly stained centrosomes was measured in dozens of control and transfected cells using the calibrated straight line tool (4.8 pixels/µm; OpenLab Version 3.0.8.) Centrosomes were classified as separated if they were >1 µm apart from one another, which occurred in only 10% of untransfected (control) cells (Fig. 6G). Transient expression of HA3-Nek2 in COS-7 cells induced separation of centrosomes in about half of the cells, consistent with a previous report (4). Expression of Myc-tagged wild-type Inh2 produced the same frequency of separated centrosomes as seen with expression of HA3-Nek2 (Fig. 6G). Truncation of Inh2 at the N terminus did not significantly reduce the ability to induce centrosome separation, whereas truncation of the C-terminal domain in Inh2-(1-118) reduced the ability to produce centrosome separation by half. The activity of these different Inh2 proteins in this assay paralleled their ability to activate Nek2 kinase in HA3-Nek2·PP1C complexes (Fig. 5B). Truncation of the IKGI sequence from the N terminus of Inh2 did not diminish its activity, whereas residues beyond position 118 were required for activation of Nek2 and centrosome separation. Consistent with the results of the binding assay, Inh2-(1-118) might bind to the Nek2·PP1C complex in cells, thereby slightly affecting the function.


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  		Fig. 6.   Effects of ectopic expression of Inh2 on centrosome structure in COS-7 cells. COS-7 cells were transfected to express HA3-Nek2, Myc-tagged wild-type Inh2 (myc-Inh2 (WT)), Inh2-(1-118), or Inh2-(14-198), and transfected cells were identified by immunofluorescence microscopy using anti-HA or anti-Myc primary antibody. Centrosomes were stained with anti-gamma -tubulin primary antibody to measure distances between centrioles in transfected cells. A-F show typical immunofluorescence images from cells expressing ectopic proteins, where the arrows indicate centrioles. Bar = 10 µm. Yellow, green, and blue indicate staining of anti-gamma -tubulin antibody, anti-Myc antibody, and anti-HA antibody and Hoechst dye, respectively. Cells (13-50) on a coverslip was scored into three groups: 1) normal, 2) dispersed centrosomes, and 3) centrosomes split >1.0 µm. The percentage of dispersed/split centrosomes is shown as bars in G. Three (Inh2-(14-197)) or four (wild-type (WT), Inh2-(1-118), and control) independent experiments were performed with each vector, and the mean values ± S.E. are indicated. *, p < 0.001; **, p < 0.01, and ***, p < 0.03 (Student's t test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

For >25 years, phosphatase Inh2 has been known to inhibit PP1C with nanomolar affinity. Biochemical studies of over a decade ago showed that Inh2 binds to monomeric PP1C and produces immediate inhibition, followed by a slower conformational inactivation of bound PP1C, a process that is reversed upon phosphorylation of a conserved Thr-Pro site in Inh2 (24). Based on these results, Inh2 has been proposed to be 1) a regulatory subunit of a cytosolic PP1C·Inh2 heterodimer referred to as MgATP-dependent phosphatase (24) or 2) a molecular chaperone for PP1C (25). More recently, it has become generally accepted that PP1C in cells is distributed among multiple regulatory subunits; however, these subunits, as well as multiple inhibitor proteins besides Inh2, compete for a common binding site on PP1C (18, 26-28). Therefore, PP1C holoenzymes exclude interaction with Inh2. The predominant idea is that PP1C binds only one protein at a time, either regulatory/targeting subunits or inhibitor proteins such as Inh2 (17). Based on the results presented here, we propose that Inh2 binds to PP1C even while PP1C is associated with Nek2. Our working hypothesis is that Inh2 functions in living cells to regulate a discrete subset of PP1 holoenzymes such as the Nek2·PP1C complex on centrosomes, wherein PP1C binds to both a regulatory subunit and Inh2 simultaneously.

Nek2 has also been recovered from a two-hybrid screen using PP1C as bait. PP1C and Nek2 were shown to associate together using the KVHF motif in Nek2 (6). In biochemical assays with purified proteins, PP1C dephosphorylated and inactivated Nek2, and Nek2 phosphorylated Thr and Ser residues in PP1Cgamma 1, which can reduce phosphatase activity. Thus, Nek2·PP1C forms a complex under double-negative feedback, with the pair exhibiting either net kinase or phosphatase activity. Modeling of similar double-negative feedback pathways predicts behavior that has been called "bistable," where there is an abrupt and complete switch between "on" and "off" states (8). This would be well suited to cell cycle events such as centrosome separation. Nek2 is tethered with centrioles via binding to a specific substrate called C-Nap1 (6, 29). Phosphorylation of C-Nap1 corresponds to loss of centrosome cohesion, and C-Nap1 is the likely substrate and effector of the Nek2·PP1C complex. In this study, we have shown that Inh2 activates the kinase activity of the Nek2·PP1C complex and that Inh2 induces centrosome separation to the same extent as expression of Nek2 itself. Furthermore, we discovered that Inh2 co-localizes with centrosomes and is concentrated in a microtubule-dependent manner to form an unusual cloud around the centrosome. Centrosomes undergo separation at mitosis; and presumably, Inh2 provides a means of transducing input from mitotic signals.

We found that interaction between Inh2 and Nek2 requires PP1C and conclude that PP1C forms a bridge binding to both proteins at the same time. Several separate subdomains in Inh2 such as the N-terminal IKGI sequence, Trp46, Thr72, and the C-terminal FEMKRKLHY sequence are thought to interact with PP1C based on assays using monomeric PP1C (15, 17, 22). Especially the N-terminal IKGI sequence of Inh2 is considered to be a primary recognition site for PP1C (15, 17). A docking site on PP1C for the IKGI subdomain in Inh2 was proposed to lie adjacent to the RVXF-binding cleft based on yeast two-hybrid analysis using various PP1C charge-to-Ala mutants (19, 20, 30). This model created the possibility that PP1C could bind to both Inh2 and a subunit with the RVXF motif at the same time. It was interesting that deletion of the N-terminal IKGI sequence from Inh2 did not fully eliminate interaction with the Nek2·PP1C complex. The rationale is that in the Nek2·PP1C complex, the docking site for RVXF on PP1C is occupied with the KVHF sequence of Nek2, so the IKGI sequence of Inh2 cannot be utilized for interaction. Instead, we found that the C-terminal domain of Inh2 between residues 119 and 198 was necessary for 1) interaction with Nek2, probably Nek2·Glc7, in yeast; 2) activation of kinase activity of the Nek2·PP1C complex; and 3) initiation of centrosome separation in fibroblasts. The Inh2-(1-118) protein bound to the Nek2·PP1C complex as well as the wild-type protein, but did not produce activation of the kinase-like wild-type Inh2 protein. Thus, the C terminus of Inh2 has a functional role in regulating the Nek2·PP1C complex at centrosomes. Presumably, docking of Nek2 induces conformational changes in PP1C to expose sites for interaction with the C-terminal subdomain of Inh2. In contrast, other regulatory subunits such as GM and MYPT1 may occlude the Inh2 site or induce a different conformation to prevent interaction of Inh2 with PP1C in these complexes.

We are intrigued by possible physiological mechanisms for regulation of the Nek2·PP1C complex via Inh2. Our results suggest that activation of Inh2 results in turning on the bistable Nek2·PP1C complex. One possibility is that the expression level of Inh2 determines the kinase/phosphatase balance of the Nek2·PP1C complex. Indeed, the expression level of Inh2 fluctuates during cell cycle progression, peaking at S phase and during mitosis (11). Ectopic expression of Inh2 did significantly increase centrosome separation. Increasing the Inh2 concentration at entry into mitosis could trigger the kinase activity of the Nek2·PP1C complex, resulting in phosphorylation of centrosomal proteins such as C-Nap1. We should not neglect the possibility of phosphorylation-induced regulation of Inh2. Centrosome separation is known to be induced in response to growth factor stimuli independent of the cell cycle (31, 32). Recently, Di Agostino et al. (33) reported that activation of Nek2 is mediated through the mitogen-activated protein kinase (MAPK) pathway in mouse spermatocytes. It has been reported that Thr72 of Inh2 can be phosphorylated by MAPK (34). In addition, cell cycle-dependent kinases such as CDK2/cyclin B and CDK5/p35 also phosphorylate Thr72 of Inh2 (35, 36). Glycogen synthase kinase-3 is also known to phosphorylate this site in Inh2. Thus, it is possible that Thr72 phosphorylation could alter the efficacy of Inh2 interaction and action with the Nek2·PP1C complex.

Binding and regulation of the Nek2·PP1C complex by Inh2 pose a new way to think about PP1 inhibitor proteins. Several lines of evidence for regulation of PP1 holoenzymes by inhibitor proteins have been accumulating. First, there was the discovery of CPI-17 (protein kinase C-potentiated inhibitor of underln]17 kDa), a myosin phosphatase inhibitor protein that inhibits either the myosin phosphatase holoenzyme or the PP1C monomer with nanomolar potency (37, 38). Clearly, this inhibitor and MYPT1 both must interact with PP1C at the same time in the same complex, probably at different sites (39). Most PP1-targeting subunits use a common RVXF sequence motif for interaction, but CPI-17 depends on phosphoryl-Thr38 for inhibition (40). Second, inhibitor-1, which does use a KIQF sequence to dock to PP1C (41), was found by two-hybrid analysis to bind directly to GADD34, which itself binds to PP1C (42). In this case, an inhibitor-1·GADD34·PP1C complex is formed. Third, another example was reported for Inh2 binding to PP1C complexes. In brain extracts, Inh2·PP1C·brain CDK5/p35 complexes were identified (36). Recently, our yeast two-hybrid screening using Inh2 as bait demonstrated interaction of Inh2 with five PP1C-binding proteins, including Nek2 (this work), spinophilin,3 and a novel Ser/Thr kinase.4 These other targets of Inh2 seemingly unrelated to mitosis might account for expression of Inh2 in somatic tissues such as skeletal muscle. In light of these developments, we propose that PP1 inhibitor proteins function as auxiliary subunits for particular PP1 holoenzymes, binding in addition to, not instead of, different regulatory subunits. In other words, the targeting subunits of PP1C also select or specify which inhibitor proteins can interact, thereby creating specific signaling pathways. A number of new inhibitor proteins for PP1 have been reported such as NIPP-1 (43), CPI-17 homologs called PHI-1 (phosphatase holoenzyme inhibitor-1) (44) and KEPI (kinase-enhanced protein phosphatase type 1 inhibitor) (45), and an ortholog of Inh2 (called inhibitor-4) (23), bringing to at least eight the number of PP1 inhibitor proteins. Learning more about the specific functions of these inhibitors is an important experimental challenge.

    ACKNOWLEDGEMENTS

We thank Professor Ian Macara and his group for plasmids and advice, Professor Shirish Shenolikar for discussions, Mary E. Foley for encouragement, Ron C. Pace for technical assistance, and Christine Palazzolo for assistance with manuscript preparation.

    FOOTNOTES

* This work was supported in part by United States Public Health Service NIGMS Grant GM56362 and NCI Grant CA40042 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by American Heart Association Mid-Atlantic Affiliate Postdoctoral Fellowship 0120538U. To whom correspondence should be addressed: Center for Cell Signaling, University of Virginia School of Medicine, P. O. Box 800577, West Complex Rm. 7225, 1400 Jefferson Park Ave., Charlottesville, VA 22908. Tel.: 434-982-0812; Fax: 434-924-1236; E-mail: me2h@virginia.edu.

Published, JBC Papers in Press, September 6, 2002, DOI 10.1074/jbc.M208035200

2 The Inh2 constructs used residues 1-197 of a total of 204 to facilitate PCR amplifications by eliminating the GC-rich coding region at the C-terminal end of the protein. The recombinant Inh2-(1-197) protein is indistinguishable in functions from the full-length protein purified from rabbit skeletal muscle and is referred to herein as the recombinant wild-type protein.

3 R. Terry-Lorenzo, D. L. Brautigan, and S. Shenolikar, submitted for publication.

4 H. Wang and D. L. Brautigan, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: CDK, cyclin-dependent kinase; PP, protein phosphatase; PP1C, protein phosphatase-1 catalytic subunit; Inh2, inhibitor-2; GST, glutathione S-transferase; HA, hemagglutinin; MOPS, 4-morpholinepropanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; MBP, myelin basic protein; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; CTD, C-terminal domain; IBF, Inh2-binding fragment; MAPK, mitogen-activated protein kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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