affinity - purified rabbit polyclonal antibody to diphosphorylated erk2   ( anti - active mapk ) was purchased from promega corp . ( madison ,   wi ) , and mouse monoclonal antibody to diphosphorylated erk2 was a   generous gift of dr . rony seger ( yung et al . , 1997 ) . in experiments performed to determine the specificity of the anti - active mapk antibody ,   wild - type or mutant ( his)6-rat erk2 ( robbins et al . , 1993 ) were expressed in bacteria , purified by ni - nitrilotriacetic acid ( nta ) metal affinity chromatography ( qiagen inc . , valencia , ca ) , and phosphorylated for 10 min at 30c with constitutively active mutant mkk1 ( g1c :   n4/s218e / s222d ; mansour et al . , 1996 ) , which was expressed in bacteria   and purified as described ( mansour et al . , 1994 ) . reactions contained 1 g   erk2 , 1 g mkk1 , 0.1 mm atp , 10 mm mgcl2 , 20 mm hepes , ph 7.4 ,   and 1 mm dithiothreitol in 25 l . alternatively , whole cell lysates were   prepared from nih 3t3 cells starved in dmem , 0% fbs overnight , and   then treated for 5 min with 10% serum and 0.1 m pma . proteins were   separated by sds - page , transferred to immobilon p ( millipore corp . ,   bedford , ma ) , reacted with primary antibody ( 1:1,000 dilution , 1 h ) followed by 0.8 g / ml goat anti  rabbit or mouse igg ( the jackson laboratories , bar harbor , me ) coupled to horseradish peroxidase , and then visualized by enhanced chemiluminiscence ( amersham life science , inc . ,   arlington heights , il ) . immunoblots were also probed using a rabbit   polyclonal antibody recognizing the cooh terminus of erk2 ( c-14 ;   santa cruz biotechnology , santa cruz , ca ) . affinity - purified rabbit polyclonal antibody to phosphorylated mkk1/2   ( anti - phosphomek1/2 ) was purchased from new england biolabs inc . wild - type ( his)6-human mkk1 was   expressed in bacteria , purified by ni - nta affinity chromatography   ( mansour et al . , 1996 ) , proteolyzed with enterokinase ( invitrogen corp . ,   carlsbad , ca ) , and phosphorylated with a constitutively active mutant of   mek kinase ( [ his]6-mekkc ; khokhlatchev et al . , 1996 this test resulted in monophosphorylated and   diphosphorylated forms of mkk1 that were subsequently resolved by   fplc using a mono q hr5/5 column equilibrated in 20 mm tris , ph 8 ,   10% ( vol / vol ) glycerol , 1 mm dithiothreitol , and were eluted with a linear   sodium chloride gradient in the same buffer . mass spectrometric peptide   mapping and sequencing were performed using a pe sciex api - iii electrospray ionization mass spectrometer ( resing and ahn , 1998 ) . this analysis revealed that the monophosphorylated form of mkk1 was modified   entirely at ser218 , indicating that phosphorylation by mekk1 was ordered under these conditions . immunoblotting using anti - phospho - mek1/2 antibody ( 1:1,000 dilution ) was performed as above . immunoblots were also probed with a rabbit polyclonal antibody recognizing   the nh2-terminus of mkk1 ( c-18 ; santa cruz biotechnology , santa   cruz , ca ) . for experiments with intact chromosomes , wild - type ( his)6-erk2 or   catalytically inactive ( his)6-erk2(k52r ) proteins were expressed in bacteria , purified by ni - nta chromatography , and phosphorylated with   constitutively active mutant mkk1 ( g7b : n4/s218d / s222d ; mansour   et al . , 1996 ) , which was expressed in bacteria and proteolyzed with enterokinase to remove the ( his)6 tag . reactions contained 170 g erk2 , 15 g   mkk1-g7b , 4 mm atp , 15 mm mgcl2 , 20 mm hepes , ph 7.4 , and 0.2%   ( vol / vol ) -mercaptoethanol in a final volume of 1 ml , and were incubated   for 3 h at 30c . erk2 was purified away from mkk and atp by adsorption to ni - nta resin incubated for 20 min at room temperature , followed by three washes with 1 ml 10 mm hepes , ph 7.4 , 0.2% -mercaptoethanol , and elution with 10 mm tris ph 8 , 0.3 m imidazole , 0.2%   -mercaptoethanol . aliquots of the activated erk2 were snap - frozen in   liquid nitrogen and stored at 80c . the reaction resulted in > 90%   diphosphorylation of erk2 at thr183 and tyr185 as assessed by mass   spectrometric peptide mapping and sequencing . the specific activity of   the wild - type phosphorylated erk2 was 1.5 mol / min / mg , measured using 0.3 mg / ml myelin basic protein ( sigma chemical co. , st . louis , mo )   and 0.1 mm atp as substrates ( mansour et al . , 1994 ) . mouse nih 3t3 , cho , or rat kangaroo ptk1 cells were grown in dmesupplemented with penicillin ( 100 u / ml ) , streptomycin ( 100 g / ml ) , and   10% fbs ( gibco - brl , gaithersburg , md ) . in some experiments , cells were treated with nocodazole ( 5 g / ml ) , for 30 min before examination . cells were grown to 75% confluence on round glass cover slips in 6-   or 10-cm plates . cover slips were rapidly rinsed in cold ( 4c ) pbs , and   were then immediately fixed under one of the following conditions : ( a )   glutaraldehyde fixation : samples were fixed with 0.1% ( vol / vol ) glutaraldehyde , 2% formaldehyde in pbs for 5 min , and were then permeabilized   with pbs , 0.1% triton x-100 for 5 min ; ( b ) neutral buffered formalin   ( nbf)/methanol fixation : samples were fixed with 10% ( vol / vol ) nbf for   5 min , and were then permeabilized with cold ( 20c ) 100% methanol   for 5 min ; ( c ) paraformaldehyde fixation : samples were fixed with 4%   ( vol / vol ) paraformaldehyde for 5 min , and were then permeabilized with   pbs , 0.1% triton x-100 for 5 min . after fixation in all cases , cells were incubated in tris - buffered saline ( tbs , 50 mm tris , ph 7.6 , 0.15 m nacl ) ,   0.1% tween 20 , 3% bsa for 1 h. coverslips were then incubated with primary antibody for 1 h , washed 4 with tbs , 0.1% tween 20 , incubated   for 1 h with fluorescein isothiocyanate- or texas red  conjugated anti - rabbit or anti - mouse secondary antibody ( 0.8 g / ml , the jackson laboratories ) , washed 4 with tbs , 0.1% tween 20 , and finally counterstained   with 4,6-diamidino-2-phenylindole ( dapi , 0.4 g / ml in pbs ) . fluorescent images were viewed and photographed using a zeiss axioplan fluorescence microscope with a photometrics sensys digital ccd camera system . controls for nonspecific staining were performed by preincubating primary antibody with 0.1 mg / ml diphosphorylated peptides cdpdhtgflt(p)ey(p)vatrwyra ( used with antibody to phosphorylated   erk ) or cvsgqlids(p)mans(p)fvgtrsy ( used with antibody to   phosphorylated mkk ) , synthesized by macromolecular resources ( ft . other primary antibodies used in immunocytochemical studies include :   the mouse monoclonal antibody 3f3/2 , which detects phosphorylated kinetochore epitopes ( 1:1,000 dilution ; cyert et al . , 1988 ) ; a rabbit polyclonal antibody recognizing the kinetochore microtubule motor , cenp - e   ( 1:200 dilution ; yen et al . , 1991 ) ; a mouse monoclonal antibody recognizing lamin b used to monitor nuclear membrane integrity ( 1:200 dilution ;   ab-1 ; calbiochem - novabiochem corp . , la jolla , ca ) , and a mouse monoclonal antibody recognizing -tubulin ( 1:10 dilution ) used to monitor spindle pole location ( neighbors et al . , mitotic cho chromosomes were isolated as described by hyman and   michison ( 1993 , except that polyamines were omitted with no difference   in results ) , and stored at 80c in pipes buffer ( 80 mm pipes , ph 7 , 1 mm   egta , 4 mm mgcl2 , 5 mm dithiothreitol ) . chromosomes were washed   by fivefold dilution into pipes buffer containing 1 mm benzamidine , 1 g/ ml aprotinin , and 0.5 m phenylmethylsulfonyl fluoride , introduced into   perfusion chambers constructed by placing glass coverslips onto two strips   of cellophane tape ( chamber volume 10 l ) , and allowed to settle by incubation on ice for 10 min ( hunt and mcintosh , 1998 ) . for immunochemistry , chromosomes were blocked by introducing 20   l of 0.2 mg / ml bsa in pipes buffer into the chamber and incubating for 2   min at room temperature . this was followed by 10 l anti - active erk   rabbit polyclonal antibody ( 1:100 dilution in pipes buffer ) or 3f3/2 monoclonal antibody ( 1:1,000 dilution in pipes buffer ) containing 0.4 g / ml   dapi ( 2 min , room temperature ) . after two washes with 30 l pipes   buffer , 20 l fluorescein isothiocyanate- or rhodamine - conjugated anti - rabbit or anti - mouse secondary antibody ( 0.8 g / ml ) were introduced and   incubated for 2 min , followed by a final wash with 30 l pipes buffer . chambers were sealed with immersion oil , and observed directly without   fixation . for experiments examining effects of erk , mkk , or protein phosphatases on immunostaining , 20 l ( 2 u ) of protein phosphatase type 2a   catalytic subunit ( pp2a ; promega corp . ) in pipes buffer was introduced   into perfusion chambers and incubated for 15 min at room temperature . this was followed by 20 l pipes buffer containing 2 m microcystin ( alexis , woburn , ma ) , and then 20 l ( 2 g ) diphosphorylated erk2-wt ,   erk2-k52r , or constitutively active mutant mkk1-g7b in 25 mm   hepes , ph 7.4 , 15 mm mgcl2 , 2 mm atp , 2 m microcystin , 1 mm   dithiothreitol . reactions were incubated for 10 min , and samples were   blocked and treated with primary and secondary antibodies as described   above . control reactions were performed by adding microcystin to pp2a   before its addition to sample , or by omitting erk or mkk from the second reaction containing mgatp . samples were photographed by immunofluorescence microscopy as   above , using a 100 objective , recording exposure times for each image . chromosomes were visualized under each condition , and the fluorescence   intensities of kinetochores were quantified by integrating absolute intensities within four pixel squares superimposed over the kinetochore pair . pixel intensities were corrected by subtracting an instrument baseline intensity of 283 intensity units , determined by photographing a coverslip   coated with uranium at varying exposures , and extrapolating to zero exposure time . affinity - purified rabbit polyclonal antibody to diphosphorylated erk2   ( anti - active mapk ) was purchased from promega corp . ( madison ,   wi ) , and mouse monoclonal antibody to diphosphorylated erk2 was a   generous gift of dr . rony seger ( yung et al . , 1997 ) . in experiments performed to determine the specificity of the anti - active mapk antibody ,   wild - type or mutant ( his)6-rat erk2 ( robbins et al . , 1993 ) were expressed in bacteria , purified by ni - nitrilotriacetic acid ( nta ) metal affinity chromatography ( qiagen inc . , valencia , ca ) , and phosphorylated for 10 min at 30c with constitutively active mutant mkk1 ( g1c :   n4/s218e / s222d ; mansour et al . , 1996 ) , which was expressed in bacteria   and purified as described ( mansour et al . , 1994 ) . reactions contained 1 g   erk2 , 1 g mkk1 , 0.1 mm atp , 10 mm mgcl2 , 20 mm hepes , ph 7.4 ,   and 1 mm dithiothreitol in 25 l . alternatively , whole cell lysates were   prepared from nih 3t3 cells starved in dmem , 0% fbs overnight , and   then treated for 5 min with 10% serum and 0.1 m pma . proteins were   separated by sds - page , transferred to immobilon p ( millipore corp . ,   bedford , ma ) , reacted with primary antibody ( 1:1,000 dilution , 1 h ) followed by 0.8 g / ml goat anti  rabbit or mouse igg ( the jackson laboratories , bar harbor , me ) coupled to horseradish peroxidase , and then visualized by enhanced chemiluminiscence ( amersham life science , inc . ,   arlington heights , il ) . immunoblots were also probed using a rabbit   polyclonal antibody recognizing the cooh terminus of erk2 ( c-14 ;   santa cruz biotechnology , santa cruz , ca ) . affinity - purified rabbit polyclonal antibody to phosphorylated mkk1/2   ( anti - phosphomek1/2 ) was purchased from new england biolabs inc . wild - type ( his)6-human mkk1 was   expressed in bacteria , purified by ni - nta affinity chromatography   ( mansour et al . , 1996 ) , proteolyzed with enterokinase ( invitrogen corp . ,   carlsbad , ca ) , and phosphorylated with a constitutively active mutant of   mek kinase ( [ his]6-mekkc ; khokhlatchev et al . , 1996 this test resulted in monophosphorylated and   diphosphorylated forms of mkk1 that were subsequently resolved by   fplc using a mono q hr5/5 column equilibrated in 20 mm tris , ph 8 ,   10% ( vol / vol ) glycerol , 1 mm dithiothreitol , and were eluted with a linear   sodium chloride gradient in the same buffer . mass spectrometric peptide   mapping and sequencing were performed using a pe sciex api - iii electrospray ionization mass spectrometer ( resing and ahn , 1998 ) . this analysis revealed that the monophosphorylated form of mkk1 was modified   entirely at ser218 , indicating that phosphorylation by mekk1 was ordered under these conditions . immunoblotting using anti - phospho - mek1/2 antibody ( 1:1,000 dilution ) was performed as above . immunoblots were also probed with a rabbit polyclonal antibody recognizing   the nh2-terminus of mkk1 ( c-18 ; santa cruz biotechnology , santa   cruz , ca ) . for experiments with intact chromosomes , wild - type ( his)6-erk2 or   catalytically inactive ( his)6-erk2(k52r ) proteins were expressed in bacteria , purified by ni - nta chromatography , and phosphorylated with   constitutively active mutant mkk1 ( g7b : n4/s218d / s222d ; mansour   et al . , 1996 ) , which was expressed in bacteria and proteolyzed with enterokinase to remove the ( his)6 tag . reactions contained 170 g erk2 , 15 g   mkk1-g7b , 4 mm atp , 15 mm mgcl2 , 20 mm hepes , ph 7.4 , and 0.2%   ( vol / vol ) -mercaptoethanol in a final volume of 1 ml , and were incubated   for 3 h at 30c . erk2 was purified away from mkk and atp by adsorption to ni - nta resin incubated for 20 min at room temperature , followed by three washes with 1 ml 10 mm hepes , ph 7.4 , 0.2% -mercaptoethanol , and elution with 10 mm tris ph 8 , 0.3 m imidazole , 0.2%   -mercaptoethanol . aliquots of the activated erk2 were snap - frozen in   liquid nitrogen and stored at 80c . the reaction resulted in > 90%   diphosphorylation of erk2 at thr183 and tyr185 as assessed by mass   spectrometric peptide mapping and sequencing . the specific activity of   the wild - type phosphorylated erk2 was 1.5 mol / min / mg , measured using 0.3 mg / ml myelin basic protein ( sigma chemical co. , st . louis , mo )   and 0.1 mm atp as substrates ( mansour et al . , 1994 ) . mouse nih 3t3 , cho , or rat kangaroo ptk1 cells were grown in dmesupplemented with penicillin ( 100 u / ml ) , streptomycin ( 100 g / ml ) , and   10% fbs ( gibco - brl , gaithersburg , md ) . in some experiments , cells   were treated with nocodazole ( 5 g / ml ) , for 30 min before examination . cells were grown to 75% confluence on round glass cover slips in 6-   or 10-cm plates . cover slips were rapidly rinsed in cold ( 4c ) pbs , and   were then immediately fixed under one of the following conditions : ( a )   glutaraldehyde fixation : samples were fixed with 0.1% ( vol / vol ) glutaraldehyde , 2% formaldehyde in pbs for 5 min , and were then permeabilized   with pbs , 0.1% triton x-100 for 5 min ; ( b ) neutral buffered formalin   ( nbf)/methanol fixation : samples were fixed with 10% ( vol / vol ) nbf for   5 min , and were then permeabilized with cold ( 20c ) 100% methanol   for 5 min ; ( c ) paraformaldehyde fixation : samples were fixed with 4%   ( vol / vol ) paraformaldehyde for 5 min , and were then permeabilized with   pbs , 0.1% triton x-100 for 5 min . after fixation in all cases , cells were incubated in tris - buffered saline ( tbs , 50 mm tris , ph 7.6 , 0.15 m nacl ) ,   0.1% tween 20 , 3% bsa for 1 h. coverslips were then incubated with primary antibody for 1 h , washed 4 with tbs , 0.1% tween 20 , incubated   for 1 h with fluorescein isothiocyanate- or texas red  conjugated anti - rabbit or anti - mouse secondary antibody ( 0.8 g / ml , the jackson laboratories ) , washed 4 with tbs , 0.1% tween 20 , and finally counterstained   with 4,6-diamidino-2-phenylindole ( dapi , 0.4 g / ml in pbs ) . fluorescent images were viewed and photographed using a zeiss axioplan fluorescence microscope with a photometrics sensys digital ccd camera system . controls for nonspecific staining were performed by preincubating primary antibody with 0.1 mg / ml diphosphorylated peptides cdpdhtgflt(p)ey(p)vatrwyra ( used with antibody to phosphorylated   erk ) or cvsgqlids(p)mans(p)fvgtrsy ( used with antibody to   phosphorylated mkk ) , synthesized by macromolecular resources ( ft . other primary antibodies used in immunocytochemical studies include :   the mouse monoclonal antibody 3f3/2 , which detects phosphorylated kinetochore epitopes ( 1:1,000 dilution ; cyert et al . , 1988 ) ; a rabbit polyclonal antibody recognizing the kinetochore microtubule motor , cenp - e   ( 1:200 dilution ; yen et al . , 1991 ) ; a mouse monoclonal antibody recognizing lamin b used to monitor nuclear membrane integrity ( 1:200 dilution ;   ab-1 ; calbiochem - novabiochem corp . , la jolla , ca ) , and a mouse monoclonal antibody recognizing -tubulin ( 1:10 dilution ) used to monitor spindle pole location ( neighbors et al . , 1988 ) . mitotic cho chromosomes were isolated as described by hyman and   michison ( 1993 , except that polyamines were omitted with no difference   in results ) , and stored at 80c in pipes buffer ( 80 mm pipes , ph 7 , 1 mm   egta , 4 mm mgcl2 , 5 mm dithiothreitol ) . chromosomes were washed   by fivefold dilution into pipes buffer containing 1 mm benzamidine , 1 g/ ml aprotinin , and 0.5 m phenylmethylsulfonyl fluoride , introduced into   perfusion chambers constructed by placing glass coverslips onto two strips   of cellophane tape ( chamber volume 10 l ) , and allowed to settle by incubation on ice for 10 min ( hunt and mcintosh , 1998 ) . for immunochemistry , chromosomes were blocked by introducing 20   l of 0.2 mg / ml bsa in pipes buffer into the chamber and incubating for 2   min at room temperature . this was followed by 10 l anti - active erk   rabbit polyclonal antibody ( 1:100 dilution in pipes buffer ) or 3f3/2 monoclonal antibody ( 1:1,000 dilution in pipes buffer ) containing 0.4 g / ml   dapi ( 2 min , room temperature ) . after two washes with 30 l pipes   buffer , 20 l fluorescein isothiocyanate- or rhodamine - conjugated anti - rabbit or anti - mouse secondary antibody ( 0.8 g / ml ) were introduced and   incubated for 2 min , followed by a final wash with 30 l pipes buffer . chambers were sealed with immersion oil , and observed directly without   fixation . for experiments examining effects of erk , mkk , or protein phosphatases on immunostaining , 20 l ( 2 u ) of protein phosphatase type 2a   catalytic subunit ( pp2a ; promega corp . ) in pipes buffer was introduced   into perfusion chambers and incubated for 15 min at room temperature . this was followed by 20 l pipes buffer containing 2 m microcystin ( alexis , woburn , ma ) , and then 20 l ( 2 g ) diphosphorylated erk2-wt ,   erk2-k52r , or constitutively active mutant mkk1-g7b in 25 mm   hepes , ph 7.4 , 15 mm mgcl2 , 2 mm atp , 2 m microcystin , 1 mm   dithiothreitol . reactions were incubated for 10 min , and samples were   blocked and treated with primary and secondary antibodies as described   above . control reactions were performed by adding microcystin to pp2a   before its addition to sample , or by omitting erk or mkk from the second reaction containing mgatp . samples were photographed by immunofluorescence microscopy as   above , using a 100 objective , recording exposure times for each image . chromosomes were visualized under each condition , and the fluorescence   intensities of kinetochores were quantified by integrating absolute intensities within four pixel squares superimposed over the kinetochore pair . pixel intensities were corrected by subtracting an instrument baseline intensity of 283 intensity units , determined by photographing a coverslip   coated with uranium at varying exposures , and extrapolating to zero exposure time . wild - type erk2 in different states of phosphorylation   and four of its mutant alleles were examined by immunoblotting in order to establish the specificity of the polyclonal   anti - active mapk antibody . reactivity was observed   with either the wild type or with a catalytically inactive   mutant ( k52r ) after their phosphorylation with active   mkk1 ( fig . 1 a , lanes 2 and 6 ) , but staining was completely absent with unphosphorylated erk2 ( fig . 1 a ,   lane 1 ) . reactivity was preferential for the diphosphorylated enzyme , since monophosphorylated erk2-y185f   or -t183a mutants were poorly recognized by the antibody ( fig . 1 a , lanes 4 and 5 ) . interestingly , antibody reactivity with monophosphorylated mutant erk2-t183e was   nearly as efficient as with diphosphorylated erk2-wt ,   indicating the importance of a negative charge in epitope   recognition ( fig . immunoblots of whole cell   extracts using the anti - active mapk antibody showed   elevated reactivity with erks 1 and 2 after stimulation of   nih 3t3 , cho , or ptk1 cells with 10% serum / phorbol ester , consistent with the expected protein kinase activation   ( fig . 1 b , top ) . importantly , no other proteins within the   extracts were strongly reactive , indicating specific recognition of erks among total cellular proteins . comparable   loading of samples was shown by immunoreactivity of the   same cell extracts using a cooh - terminal erk2 antibody ( fig . immunocytochemical staining of the anti - active   mapk antibody was examined in nih 3t3 cells in various   stages of mitosis . cells in interphase showed little or no reactivity , but marked increases in active erk staining were   observed in the nuclei of prophase cells identified by the   morphology of their chromatin ( fig . 2 , a and g ) . costaining of lamin b showed that the increased erk reactivity   during prophase occurred before nuclear envelope breakdown ( fig . 3 ) . cells in metaphase showed a distinct localization of active erk to regions surrounding the condensed chromosomes ( fig . close comparison   with dapi staining indicated that active erk staining   mainly occurred in regions surrounding chromatin , while   condensed chromosome arms were not as strongly reactive . 2 , h and i ) whose location was confirmed by   costaining with -tubulin antibodies ( data not shown ) . in   anaphase , active erk immunoreactivity was more diffusely localized and largely excluded from chromosomes   ( fig . / cytokinesis , active erk   appeared localized within cytoplasmic regions of both   daughter cells as well as the midbody ( fig . the specificity of immunostaining was also tested by   preincubating the primary antibody with a diphosphorylated peptide corresponding to the phosphoepitope on   erk . with phosphopeptide competition , immunoreactivity was also absent when the   primary antibody was omitted ( data not shown ) . experiments were also performed using a monoclonal antibody   to active diphosphorylated erk , and showed identical   staining patterns ( data not shown ) . finally , experiments   were performed using three different fixation protocols ,   including 0.1% glutaraldehyde/2% formaldehyde , 4% paraformaldehyde , or 10% neutral buffered formalin / methanol . identical results were observed in all cases ( data not   shown ) , indicating that the cellular localization of active   erk during mitosis is not a fixation artefact . localization of the upstream kinase , mkk , was examined using the anti - phospho - mek1/2 antibody that is selective for active forms of mkk1 and 2 . the specificity of   this antibody was tested by immunoblotting mkk1 that   had been phosphorylated in vitro with mekk1 and purified by mono - q fplc . this antibody reacted with both   mono- and diphosphorylated mkk1 , with preference for   the diphosphorylated form , while no reactivity was observed with unphosphorylated mkk1 ( fig . 4 a , lanes   14 ) . monophosphorylated mkk1 has measurable activity that is 25% that of diphosphorylated enzyme ( resing   et al . , 1995 ) . therefore , this antibody distinguishes between active and inactive forms of mkk . unlike the anti - active erk antibody , the anti - phospho - mek1/2 antibody did not recognize a mkk mutant containing acidic   amino acid substitutions at phosphorylation sites ( g1c ;   fig . immunoblots of whole cell extracts   showed elevated reactivity with mkks 1 and 2 after stimulation of nih 3t3 , cho , or ptk1 cells , but little or no reactivity with other cellular proteins ( fig . whole   cell extracts were also immunoblotted using an antibody   recognizing the nh2 terminus of mkk1 to demonstrate   comparable loading ( fig . immunocytochemical staining of nih 3t3 cells with the   anti - phosphomek1/2 antibody showed little reactivity   with interphase cells , but nuclear staining of active mkk   increased significantly during prophase ( fig . 5 , a and g ) . this result is notable because it represents the only case in   which nuclear localization of mkk has been observed under normal cellular conditions without perturbing nuclear   export ( fukuda et al . , 1996 ; fukuda et al . localization of active mkk   was apparent at spindle poles during metaphase and   anaphase ( fig . 5 , b , c , h , and i ) and at the midbody during telophase / cytokinesis ( fig . unlike active   erk , however , active mkk appeared more diffusely localized throughout metaphase cells , and largely excluded   from chromosomes ( fig . antibody preincubation with a diphosphorylated peptide corresponding to   the phosphoepitope on mkk1/2 eliminated staining of mitotic cells ( fig . 5 , e , f , k , and l ) , supporting the specificity of immunoreactivity with this antibody .   to visualize the nuclear localization of active erk in   more detail , we examined ptk1 cells , which have fewer   chromosomes and a flatter morphology during mitosis . as   observed with nih 3t3 cells , active erk staining was absent in interphase ptk1 cells ( data not shown ) , but increased in nuclei during prophase ( fig . spindle pole localization of active erk was also observed   during prophase , before nuclear envelope breakdown   ( fig . the higher resolution available from images of   ptk1 cells showed that much of the active erk staining   was peripheral to prophase chromosomes . furthermore , a   staining pattern of paired dots could be observed on each   prophase chromosome , suggesting a specific localization   of active erk to kinetochores . this pattern was confirmed by colocalization of active erk with the microtubule motor cenp - e ( fig . 7 , c and d ) , which localizes to   kinetochores in vivo ( yen et al . , inspection of cells   at different stages in prophase to prometaphase suggested   a temporal order of association : active erk appears at kinetochores earlier in prophase than cenp - e , and leaves   the kinetochore earlier in metaphase / anaphase ( fig . the kinetochore localization of active erk that was so   evident during prophase and prometaphase was more difficult to identify on chromosomes aligned at the metaphase plate , and was completely absent during anaphase   ( fig . in contrast , cenp - e staining was still apparent at kinetochores during anaphase   ( fig . 7 f ) . these results suggest that either the phosphorylation or the targeting of erk to this mechanically important region decreases as chromosomes separate . with   phosphopeptide competition , active erk immunoreactivity was undetectable in mitotic ptk1 cells ( data not   shown ) . ptk1 staining was also examined using an antibody recognizing the cooh terminus of erk2 , which is not selective for the protein 's state of phosphorylation . , erk was primarily localized in cytoplasmic pools as previously reported ( lenormand et al . ,   1993 ) , but in prophase , nuclear staining increased significantly , indicating the relocalization of a significant fraction   of the total erk pool ( fig . however , in contrast to active erk , kinetochore localization within the total pool of   nuclear erk was masked , indicating that the active enzyme associating with kinetochores represents a small   fraction of total erk . although kinetochore staining of active erk was not   apparent in fixed and permeabilized nih 3t3 cells ( fig . 2 ) , we were also unable to observe cenp - e staining , indicating that kinetochores were refractory to staining in   these experiments , perhaps due to cell shape . consistent   with this finding , colocalization of active erk and cenp - e   at kinetochores was observed in porcine kidney epithelial   llcpk1 cells , which also exhibit a flat morphology during   mitosis ( data not shown ) . as noted below , kinetochore localization of active erk was also observed on isolated   chromosomes from nih 3t3 cells , cho cells , and 9948   human lymphoblasts . active erk localized to kinetochores   of every cell type we examined , indicating that it represents a general phenomenon in mammalian cells . examination of phospho - mkk in ptk1 cells also showed   increased nuclear staining during prophase , with spindle   pole localization during prophase to anaphase , and was   mostly excluded from chromosome arms ( fig . in general , phospho - mkk showed infrequent reactivity   with kinetochores compared with phospho - erk , although   paired dot patterns were evident in some cases ( fig . these data indicate that active erk and active mkk accumulate in nuclei during prophase , and suggest that active erk might preferentially associate with kinetochores . these results suggest that erk phosphorylation might   serve as a signal for chromosome targeting . indeed , the temporal pattern of phosphoerk association with kinetochores is similar to that observed using   3f3/2 , a monoclonal antibody raised to crude maturation   promoting factor ( mpf ) preparations that recognizes nuclear phosphorylated antigens ( cyert et al . , 1988 ; gorbsky   and ricketts , 1993 ) . kinetochore phosphoepitopes have   been shown to react with this antibody under conditions   where chromosomes are misaligned or detached from microtubules , suggesting that the decreases in antigen reactivity that occur during mitotic progression are caused by   dephosphorylation as the chromosomes align under microtubule - induced tension at the metaphase plate ( gorbsky and ricketts , 1993 ; nicklas et al . , 1995 ) . temporal correlations in mitotic localizations of active   erk and the 3f3/2 phosphoepitope were observed by   double immunofluorescence staining of ptk1 cells . kinetochore colocalization of both antigens was apparent from   early prophase through prometaphase ( fig . could sometimes be observed with anti - active mapk antibody ,   but never with 3f3/2 antibody ( fig . 10 , e and f ) , suggesting that the dephosphorylation of the 3f3/2 antigen during   metaphase precedes inactivation of erk . in both cases , treatment of cells with nocodazole to disrupt spindle microtubules resulted in stabilized   kinetochore localization of active erk as well as the 3f3/2   antigen ( fig . untreated metaphase cells   were inspected for chromosomes that were aberrantly displaced from the metaphase plate , which is easily identifiable by dapi staining . in several such examples , kinetochores on displaced chromosomes showed significantly   enhanced staining with anti - active mapk antibody   compared with the reduced staining of aligned chromosomes ( fig . thus , the activity of erks   bound to kinetochores appears to correlate inversely with   kinetochore - microtubule attachments , consistent with the   behavior of the 3f3/2 phosphoantigen , as reported previously ( gorbsky and ricketts , 1993 ; nicklas et al . , 1995 ) .    to explore a possible functional relationship between   active erk and the 3f3/2 antigen , chromosomes were isolated from cho cells , immobilized on coverslips within   perfusion chambers , and visualized by immunofluorescence . staining on   chromosome arms was visible , but low compared with kinetochore staining , indicating that the most stable association is at the kinetochores . similar results were observed   with isolated chromosomes from nih 3t3 cells or 9948   lymphoblasts ( data not shown ) . after perfusion with pp2a ,   the fluorescence staining for 3f3/2 antigens at kinetochores of cho cells was dramatically decreased ( fig . at least some of this activity returned after treating   phosphatase - treated chromosomes with microcystin , active diphosphorylated erk2 , and mg / atp ( fig . these results motivated an optical quantification of   epitope levels after various experimental treatments . quantitative measurements showed decreased staining of   kinetochores with anti - active mapk antibody in response to phosphatase treatment ( fig . 13 a ) , conditions   where dephosphorylation of erk would be expected . in   addition , the fluorescence intensity of 3f3/2 antibody   staining at kinetochores decreased by 75% after 15 min of   reaction with pp2a ( fig . 13 , b and c ) , as compared with   controls in which pp2a was omitted or dephosphorylation   was blocked with microcystin ( fig . this result is consistent with the proposed recognition of a phosphorylated epitope by the 3f3/2 antibody ( cyert et al . after pp2a treatment , microcystin was added to block   dephosphorylation , and purified diphosphorylated erk2   was perfused into chambers along with mgatp . in this   case , the fluorescence intensity of 3f3/2 staining increased   by four to fivefold after 10 min of reaction with wild - type   active erk2 ( fig . the staining also increased by about twofold when chromosomes were perfused with mgatp alone , or when perfused with mgatp   in presence of diphosphorylated , catalytically inactive mutant erk2-k52r . furthermore , perfusion of chromosomes with constitutively active mutant mkk1-g7b in the   presence of mgatp after pp2a treatment led to a significant increase in 3f3/2 staining at kinetochores compared   with perfusion with mgatp alone ( fig . 13 immunoblotting of active erk showed no reactivity   with 3f3/2 antibody ( data not shown ) , indicating that   diphosphorylated erk is not likely to be the 3f3/2 reactive phosphoantigen . taken together , our results are consistent with a model in which the 3f3/2 antigen can be   phosphorylated in response to addition of exogeneous   erk2 , as well as by endogenous protein kinases located at   kinetochores , possibly including erk2 . these findings indicate that the 3f3/2 phosphoepitope may be regulated by   erk , suggesting a role for erk function at kinetochores   in mediating responses to the microtubule attachments   that control anaphase onset . the results presented above suggest that active forms of   both erk and mkk become concentrated in the prophase nuclei of somatic mammalian cells . it becomes clear at prometaphase and is maintained through anaphase , but the strength of erk   localization at kinetochores decreases as the chromosomes attach to the spindle and become organized at the   metaphase plate . this behavior is reminiscent of the staining seen with 3f3/2 , a monoclonal antibody that recognizes a phosphoepitope found on mature but unattached   chromosomes . the intensity of 3f3/2 staining of kinetochores on isolated mammalian chromosomes treated with   phosphatases and kinases is consistent with the hypothesis   that erk contributes to the formation of the 3f3/2   epitope , and therefore may play a role in regulating kinetochore function during mitosis . our results with antibodies selective for the active phosphorylated forms of erk and mkk demonstrate significant increases in the specific localizations of the endogeneous enzymes during mammalian cell mitosis . the   concentration of these enzymes in nuclei during early   prophase of cycling somatic cells supports a role for the   map kinase pathway in regulating early mitotic events . this result was unexpected because previous biochemical   studies of kinase activity measured by sds - page gel mobility retardation or in - gel phosphorylation assays of mammalian cell extracts failed to show erk activation during   mitosis ( tamemoto et al . , 1992 ; edelmann et al . , 1996 ) .   these differences can be reconciled by comparing the   staining patterns seen with the anti - active mapk antibody versus staining with an antibody recognizing both   unphosphorylated and phosphorylated erk . the mitotically active pool of erk appearing at discrete mitotic locations represents a minor fraction of the total erk , and this highlights the key advantage of antibodies that   selectively recognize active phosphorylated forms of regulatable enzymes for studies of cell process control . a requirement for erk in m phase entry has been extensively studied in several model systems for meiotic cell   division ( reviewed by yew et al . , erk activation promotes release   from g2 arrest and entry into metaphase i. in dividing somatic cells , activation of erk by active mutant mkk expression enhances cell growth rate , consistent with a role   in promoting cell cycle progression ( seger et al . , 1994 ) . significantly , recent findings suggest a requirement for   mkk and erk during the g2-m transition in cycling   mammalian cells ( j.h . synchronized nih 3t3   cells allowed to progress through s phase were retarded in   m phase entry after inhibition of mkk / erk with a dominant negative mutant mkk1 or the mkk1 inhibitor ,   pd98059 . under these conditions , tyrosine dephosphorylation of cdc2 was suppressed , indicating that mkk / erk   is somehow involved in mitotic entry upstream of cyclin   b / cdc2 activation . these findings are consistent with our   results showing that mkk and erk are activated early in   prophase before nuclear envelope breakdown . erk also prevents release from metaphase ii arrest in   oocytes through inhibition of cyclin b degradation and stabilization of cdc2 activity ( sagata , 1997 ) . injection of mos ,   active mkk , or active erk into two - cell frog embryos induces a similar m phase arrest with elevated cyclin b / cdc2   activity ( haccard et al . , 1993 ; kosako et al . , similar   findings were reported in frog embryo extracts , where g2   arrest as well as m phase arrest were observed in response   to mos , mkk , or erk , depending on the timing of addition ( abrieu et al . , 1997 ; walter et al . , 1997 ) . these results   may be important in understanding erk and mechanisms   for spindle checkpoint regulation , given that mitotic frog   extracts undergo metaphase arrest when challenged with   high levels of sperm dna together with microtubule - destabilizing agents in a manner that depends upon elevated erk activity and stabilization of cyclin b / cdc2 activity ( minshull et al . these   data suggest that erk promotes metaphase spindle   checkpoint arrest by interfering with the activation of the   anaphase - promoting complex ( apc ) , perhaps by functioning upstream or in parallel with other checkpoint regulatory pathways ( abrieu et al . , 1996 ) . the kinetochore localization of active erk that we observe in ptk1 cells suggests a mechanism by which erk   might regulate anaphase entry . previous findings indicate   that microtubule tension on chromosomes and kinetochore attachment is an important feature of the mitotic   checkpoint regulating anaphase entry ( reviewed by nicklas , 1997 ) . in particular , it has been suggested that dominant signals derived from unattached kinetochores may   delay anaphase entry ( mcintosh , 1991 ) . such signals may   involve phosphorylation of proteins within pathways that   regulate apc activation and cyclin b degradation , which   are required for the metaphase - to - anaphase transition   ( gorbsky , 1997 ; nigg et al . , 1996 ) . a useful probe for   phosphorylation signals has been the 3f3/2 monoclonal   antibody , originally raised to impure mpf preparations ,   which appears to recognize phosphorylated antigens located at kinetochores . reactivity with kinetochores is high   during prophase and prometaphase , and appears to diminish during metaphase as microtubules attach . on the other   hand , reactivity increases during kinetochore detachment ,   and loss of tension induced in response to nocodazole or   by physical misalignment of chromosomes away from the   metaphase plate ( gorbsky and ricketts , 1993 ; nicklas et   al . , 1995 ) . furthermore , 3f3/2 antibody microinjection   into cycling cells delays anaphase onset , suggesting that   dephosphorylation of this antigen is a necessary event for   chromosome segregation ( campbell and gorbsky , 1995 ) . we observed that the appearance and disappearance of   active erk at kinetochores resembled the timing and behavior of 3f3/2 immunoreactivity previously reported ,   suggesting that regulation of erk may be linked somehow   to 3f3/2 antigen phosphorylation . active erk appeared at   kinetochores during prophase and prometaphase and   disappeared at some point during the metaphase - to - anaphase transition . furthermore , stable kinetochore staining was observed under conditions of microtubule disruption or on chromosomes that were displaced from the   metaphase plate . isolated chromosomes provided an excellent model system to test whether kinetochore 3f3/2   antigens could be modulated by erk activity , allowing   quantification of immunostaining after direct reactions   with added enzymes . interestingly , both active erk and   the 3f3/2 antigen were present on kinetochores , indicating a stable association of these antigens with isolated   chromosomes . reduced staining of each was observed   after protein phosphatase treatment , most likely due to   phosphoepitope dephosphorylation . importantly , the enhanced reactivity of kinetochores with 3f3/2 antibody after incubation with active erk2 or active mkk1 indicates   that 3f3/2 antigen(s ) are likely to be targets for erk or   erk - regulated kinases . the only known substrates for   mkk1 are erks 1 and 2 , suggesting that the 3f3/2 antigen is phosphorylated by erk or an erk - regulated kinase , endogeneously localized at kinetochores . it is therefore reasonable that erk in its active form might serve as   a signal at kinetochores , allowing cells to sense improper   chromosome attachment to spindle microtubules via phosphorylation of the 3f3/2 antigen . dephosphorylation of   the 3f3/2 antigen after microtubule attachment and resulting increased tension at kinetochores might then release   the inhibitory checkpoint machinery , facilitating apc activation and protein degradation events needed for anaphase entry . the p38 mapk has been   implicated in spindle assembly checkpoint and mitotic arrest in mammalian cells ( takenaka et al . , 1998 ) , based on   data reporting the activation of p38 mapk during nocodazole - induced mitotic arrest and partial suppression of   the nocodazole - induced checkpoint by a p38 mapk inhibitor . so far we have found no evidence for p38 mapk   activation in mitotic cells , localization of active enzyme at   kinetochores of permeabilized cells , or isolated chromosomes using an antibody from new england biolabs ( beverly , ma ) that recognizes active , diphosphorylated p38   mapk ( data not shown ) . the specificity of this antibody   was confirmed by enhanced immunofluorescence of permeabilized cells or reactivity in immunoblots of cells exposed to uv irradiation ( data not shown ) . another important result from our study is the localization of active erk at the chromosome periphery during   prophase and prometaphase , as well as at spindle poles   during prophase , metaphase , and anaphase . the pattern of   active erk on spindle microtubules , spindle poles , and kinetochores suggests a role of erk in regulating spindle   assembly , possibly through regulation of motor activity or   microtubule polymerization . spindle pole immunostaining   was previously reported during meiotic metaphase in   mouse oocytes and mitosis in xenopus tadpole cells ( verlhac et al . , 1993 ; wang et al . , 1997 ) . erk staining of microtubules has also been observed in nih 3t3 cells , though it   is somewhat dependent on the antibody used ( rezka et   al . , 1995 ) , and association of active and inactive erk   pools with tubulin and maps has been demonstrated biochemically ( morishima - kawashima and kosik , 1996 ; mandelkow et al . , 1992 ) . furthermore , adding active mapk to   interphase frog embryo extracts leads to microtubule   shortening and organization , which resembles a mitotic array ( gotoh et al . , 1991 ) . potential mediators of microtubule depolymerization include maps such as tau , which ,   upon phosphorylation by erk enhances the probability   of microtubule depolymerization ( shiina et al . , 1992 ;   hoshi et al . , 1992 ; dreschsel et al . , another possible   target is stathmin / op18 , which facilitates microtubule catastrophe and mitotic destabilization of interphase microtubules ( belmont and mitchison , 1996 ) , and is hyperphosphorylated during mitosis at sites targeted by several   kinases , including erk ( brattsand et al . , 1994 ; marklund   et al . , 1993 ; larsson et al . , 1995 ) . in summary , current data suggest that the mkk / erk   pathway plays multiple roles during mitosis , promoting   critical events that regulate mitotic entry or spindle assembly and function , as well as inhibiting events needed for   anaphase entry . our findings raise new questions about   downstream targets for erk at different stages of mitotic   progression , and the potential overlap in specificity between erk and cyclin b / cdc2 for substrates . further questions are also raised regarding the mechanism of mkk   and erk activation in g2 . it is reasonable to suggest that   this regulatory pathway involves at least some of the components identified as upstream regulators in g1 . the mitotic activation of mkk and its requirement for mitotic   entry in nih 3t3 cells ( j.h . wright and e.g. krebs , personal communication ) is consistent with previous reports   of mitotic activation of src and raf as well as a requirement for src activation during m phase entry ( roche et   al . at this time , no evidence exists for a mitotic role for ras that may explain src or raf   regulation . therefore , the possibility exists that alternative   mechanisms , perhaps involving novel upstream regulators   of the mapk pathway , function to control m phase entry .   finally , an important event revealed in our studies is the   nuclear uptake of both mkk and erk during prophase ,   before nuclear envelope breakdown . although previous   work has demonstrated erk translocation after activation in response to growth factors ( lenormand , 1993 ) , nuclear localization of mkk has been demonstrated only   with mkk mutants lacking its nuclear export signal or under conditions where nuclear export is inhibited ( fukuda ,   1996 ; fukuda , 1997 ; jaaro et al . , 1997 ) . localization of active mkk during prophase identifies a unique physiological condition where nuclear uptake is favored , and specifies an important function of regulatory mechanisms for   nuclear import during cell cycle progression . ( a ) immunoblots of wild type or mutant recombinant erk2 phosphorylated with constitutively active mutant mkk1-g1c . ( bottom )   reactivity with antibody recognizing the cooh terminus of   erk2 , where erk2 gel mobility is retarded upon phosphorylation . ( b ) immunoblots of whole cell extracts ( 20 g ) from nih   3t3 , cho , and ptk1 cells that were serum - depleted overnight ,   and then treated with or without 10% serum + 0.1 m pma for   5 min ( pma ) . ( top ) specific immunoreactivity of anti - active   mapk antibody with phosphorylated erk1 and erk2 ( arrows ) . only phosphorylated erk2 was observed in cho cell lysates . ( bottom ) corresponding levels of erk2 , probed with an   antibody recognizing the cooh terminus of erk2 . filled arrows show phosphorylated erk2 with retarded mobility , and   open arrows show unphosphorylated erk2 cells were   fixed with glutaraldehyde , and were then stained with dapi ( a f  ) and anti - active mapk antibody probed with texas red conjugated secondary antibody ( g  l ) . fluorescence of representative cells in ( a and g ) interphase ( upper cells ) and prophase   ( lower cell ) ; ( b and h ) metaphase ; ( c and i ) anaphase ; and ( d   and j ) telophase are shown . note active erk staining at spindle   poles in metaphase and anaphase ( h and i ) . ( e , f , k , and l ) in peptide competition controls , anti - active mapk antibody was preincubated with 0.1 mg / ml   phosphopeptide before incubation with coverslips . under these   conditions , mitotic cells showed no erk immunoreactivity , as   demonstrated with prophase ( e and f ) and metaphase ( k and l )   cells . nih 3t3 cells were fixed with   glutaraldehyde , and were   then stained with dapi ( a   and d ) and costained with ( b   and e ) anti - active mapk   antibodies probed with texas   red  coupled secondary antibodies or ( c and f ) lamin b   antibodies probed with fluorescein isothiocyanate - conjugated secondary antibodies . patterns of lamin b staining   indicate that nuclear envelope membranes are intact in   prophase cells during erk   activation . corresponding interphase cells are shown to   the left of each prophase cell   in the two representative images . ( a ) immunoblots of wild - type or mutant recombinant mkk1 , after   phosphorylation with constitutively active mutant mekkc . differences in gel mobility are a result of removing the   5-kd nh2 terminal ( his)6 tag from mono- or diphosphorylated   mkk1 , and deleting residues 4451 in mkk1-g1c . ( b ) immunoblots of whole cell extracts ( 20 g ) from nih 3t3 , cho , and   ptk1 cells that were serum - depleted overnight , and then treated   with or without 10% serum + 0.1 m pma for 30 min . ( top )   specific immunoreactivity of anti - phosphomek1/2 antibody with   phosphorylated mkk1 and mkk2 , indicated by arrows . ( bottom ) corresponding levels of mkk1 probed with an antibody   recognizing the nh2 terminus of mkk1 . cells were   fixed with nbf / methanol , and were then stained with dapi ( a f ) and anti - phosphomek1/2 antibody probed with texas red conjugated secondary antibody ( f  l ) . fluorescence of representative cells in ( a and g ) interphase and prophase ; ( b and h )   metaphase ; ( c and i ) anaphase ; and ( d and j ) telophase are   shown . ( e , f , k , and l ) in peptide competition controls , anti - phosphomek1/2 antibody was preincubated with 0.1 mg / ml phosphopeptide before incubation with   coverslips . under these conditions , mitotic cells showed no active   mkk immunoreactivity as demonstrated with prophase ( e and   f ) and metaphase ( k and l ) cells . cells were   fixed with glutaraldehyde , and were then stained with dapi and   anti - active mapk antibody probed with texas red  conjugated secondary antibody . fluorescence of representative cells in   ( a ) early prophase ; ( b ) late prophase ; ( c ) prometaphase ; ( d )   metaphase ; ( e ) anaphase ; and ( f ) telophase . interphase cells are   also shown in e and f. staining patterns were also reproduced   with paraformaldehyde or nbf / methanol fixation . controls   show no immunoreactivity when anti - active mapk antibody   was preincubated with 0.1 mg / ml phosphopeptide before incubation with coverslips ( data not shown ) . ptk1   cells were fixed with glutaraldehyde , and were then stained with   dapi together with mouse monoclonal anti - phosphomapk antibody and rabbit anti - cenp - e antibody , probed with texas   red or fluorescein isothiocyanate fluorescence of representative cells in ( a   and b ) prophase ; ( c and d ) prometaphase ; and ( e and f )   anaphase ; showing kinetochore staining of active erk early in   prophase , before the appearance of cenp - e , and disappearance   of kinetochore - associated active erk during anaphase before   the disappearance of cenp - e . an interphase cell is also shown   in e and f. active erk localized to kinetochores is a minor fraction of the total erk pool . ptk1 cells were fixed with glutaraldehyde , and were then stained with ( a ) cooh - terminal erk2 antibody probed with texas red  conjugated secondary antibody ;   or ( b ) dapi . although a significant   percentage of erk is nuclear in prophase , kinetochore localization can not be detected . cells were   fixed with glutaraldehyde , and were then stained with dapi and   anti - phosphomek1/2 antibody probed with texas red  conjugated secondary antibody . fluorescence of representative cells in   ( a ) prophase ; ( b ) prometaphase ; ( c ) metaphase ; and ( d )   anaphase . controls show no immunoreactivity when anti - phosphomek1/2 antibody was preincubated   with 0.1 mg / ml phosphopeptide before incubation with coverslips   ( data not shown ) . ptk1 cells were fixed with glutaraldehyde , and were then   stained with dapi , together with anti - active mapk antibody   probed with texas red  coupled secondary antibody and 3f3/2   antibody probed with fluorescein isothiocyanate - conjugated secondary antibody . fluorescence of representative cells in ( a and   b ) prophase ; ( c and d ) prometaphase ; and ( e and f ) metaphase ,   showing corresponding appearances of active erk and 3f3/2 antigen at kinetochores early in prophase , and disappearance at or   after metaphase . ( a   and b ) ptk1 cells were treated with nocodazole for 30 min , and   were then fixed with glutaraldehyde and stained with dapi , anti - active mapk antibody , and 3f3/2 antibody as in fig . 10 . fluorescence of representative cells in metaphase showed stable kinetochore staining of ( a ) active erk and ( b ) 3f3/2 antigen . ( c   and d ) untreated ptk1 cells fixed and stained with dapi or anti - active mapk antibody . high immunoreactivity of active   erk is observed on metaphase chromosomes that are misaligned   from the metaphase plate . ( a and b ) isolated cho cell   chromosomes bound to glass coverslips were stained without fixation with dapi and ( a ) anti - active mapk antibody or ( b )   3f3/2 antibody , probed in each case with texas red  conjugated   secondary antibodies . ( c and d ) isolated chromosomes were incubated with added ( c ) pp2a for 15 min , or ( d ) pp2a for 15   min , followed by 2 m microcystin , 2 g active erk2 , and   mgatp for 10 min . chromosomes were then stained with dapi   and 3f3/2 antibody , followed immediately by data collection . sensitivity of active erk or 3f3/2 antigen at isolated   kinetochores to dephosphorylation or rephosphorylation by   added enzymes . ( a ) isolated chromosome samples bound to   glass coverslips were incubated without or with pp2a ( 2u ) for 15   min , followed by staining with dapi and anti - active mapk   antibody , as in fig . ( b ) chromosomes were incubated without or with   pp2a for 15 min , with pp2a followed by 2 m microcystin , 2 g   active erk2 , and mgatp , or with pp2a followed by microcystin and mgatp for 10 min . ( c ) chromosomes were incubated with   pp2a+microcystin for 15 min , with pp2a for 15 min minus inhibitor , with pp2a for 15 min followed by microcystin , active   erk2 , and mgatp , or with pp2a for 15 min followed by microcystin , diphosphorylated catalytically inactive erk2-k52r , and   mgatp . ( d ) chromosomes were incubated with pp2a for 15 min minus   inhibitor followed by microcystin and mgatp or microcystin , active mkk1-g7b , and mgatp for 15 min . average fluorescence intensities and standard errors are shown , normalized to controls in   each experiment .
the mitogen - activated protein ( map ) kinase pathway , which includes extracellular signal  regulated protein kinases 1 and 2 ( erk1 , erk2 ) and map   kinase kinases 1 and 2 ( mkk1 , mkk2 ) , is well - known   to be required for cell cycle progression from g1 to s   phase , but its role in somatic cell mitosis has not been   clearly established . 
 we have examined the regulation   of erk and mkk in mammalian cells during mitosis   using antibodies selective for active phosphorylated   forms of these enzymes . in nih 
 3t3 cells , both erk   and mkk are activated within the nucleus during early   prophase ; they localize to spindle poles between   prophase and anaphase , and to the midbody during cytokinesis . during metaphase 
 , active erk is localized in   the chromosome periphery , in contrast to active mkk ,   which shows clear chromosome exclusion . 
 prophase activation and spindle pole localization of active erk   and mkk are also observed in ptk1 cells . 
 discrete localization of active erk at kinetochores is apparent by   early prophase and during prometaphase with decreased staining on chromosomes aligned at the   metaphase plate . 
 the kinetochores of chromosomes   displaced from the metaphase plate , or in microtubule - disrupted cells , still react strongly with the active erk   antibody . 
 this pattern resembles that reported for the   3f3/2 monoclonal antibody , which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles , and has been implicated in the mitotic checkpoint for anaphase onset ( gorbsky and   ricketts , 1993 . 
 j. cell biol . 
 122:13111321 ) . 
 the 3f3/2   reactivity of kinetochores on isolated chromosomes decreases after dephosphorylation with protein phosphatase , and then increases after subsequent phosphorylation by purified active erk or active mkk . 
 these results suggest that the map kinase pathway has   multiple functions during mitosis , helping to promote   mitotic entry as well as targeting proteins that mediate   mitotic progression in response to kinetochore attachment .