Patent Application: US-84324501-A

Abstract:
endophilin i is a brain - specific protein functioning in clathrin - mediated endocytosis . the present invention is based on the finding that the rat germinal center kinase - like kinase , a member of the germinal center kinase family of c - jun n - terminal kinase activating enzymes , is a novel endophilin i - binding partner . in a first aspect of the present invention , the novel interaction between endophilin i and rglk is put to use in a novel screening assay . in a second aspect of the present invention , the interaction between endophilin i and glk is modulated for therapeutic purposes , namely for the prevention and / or curtailment of neurological disorders associated with the jnk pathway . jnk - mediated neuronal cell death is believed to play an important role in injuries and diseases involving neuronal degeneration , such as huntington &# 39 ; s disease .

Description:
the present invention relates to the discovery of a novel sh3 domain - mediated protein - protein interaction between the endophilin protein family and the protein kinase , germinal center kinase - like kinase ( glk ). the sites of interaction have been identified for both proteins . the ability of endophilin to regulate the activity of glk has also been established . moreover , an antibody has been raised against glk and used to identify that glk is expressed in specific neuronal populations in the brain . rglk is also detected in glial cells in hippocampal cells or cortical cells in culture . this interaction is likely to be important in glk - mediated jnk activation in cells of the nervous system and may be directly involved in their apoptotic pathways . additionally , there is supplementary evidence for a role of glk - endophilin interactions in jnk - mediated neuronal degeneration , specifically in huntington &# 39 ; s disease . to better understand interactions mediated by endophilin i , we screened a rat brain expression library with the endophilin i sh3 domain . we identified synaptojanin 1 and dynamin i , and surprisingly , we also identified and cloned a rat homologue of the human germinal center kinase - like kinase ( hglk ). hglk was originally identified based on its homology to members of the germinal center kinase ( gck ) family of protein kinases ( diener et al ., 1997 ) and is a group i gck ( kyriakis , 1999 ). group i gcks are mitogen - activated protein kinase ( mapk ) kinase kinase kinases ( map4ks ) that function upstream of c - jun n - terminal kinase ( jnk ) in a variety of cell types and are activated in response to environmental stress , treatment with inflammatory mediators of the tnf family , and the vascular responses to ischemia ( kyriakis , 1999 ). group i gcks are composed of an n - terminal kinase domain and a c - terminal regulatory domain with multiple sh3 domain consensus - binding sites ( kyriakis , 1999 ). the mechanisms by which events at the cell surface are linked to activation of glk and other gcks , and the role of sh3 domain - mediated interactions in these processes remain poorly understood . the identification of rat glk ( rglk ) as an endophilin i - binding partner suggests a role for endophilin i in the regulation of glk function . to explore this , we first confirmed the rglk - endophilin i interaction in multiple systems in vitro and used co - immunoprecipitation analysis from transfected cells and brain extracts to demonstrate the interaction in vivo . in fact , overlay assays suggest that endophilin i is the major sh3 domain - binding partner for rglk in brain . importantly , we have found that endophilin i functions directly in glk - mediate jnk activation . unlike most gck family members , rglk is expressed almost exclusively in brain where it is detected in glial cells and in neuronal populations that are known to undergo jnk - mediated apoptotic cell death in different models of neuronal stress and disease . taken together , these data implicate endophilin i in the activation of jnk and provide evidence of a novel link between the endocytic machinery and the jnk signaling pathway . an oligo ( dt ) primed λzap ii rat brain cdna expression library , size selected for clones greater than 4 kb ( snutch et al ., 1990 ; gift from dr . terry snutch , university of british columbia ), was plated at 20 , 000 plaque forming units per 150 mm 2 plate . protein expression was induced using isopropyl - β - d - thiogalactopyranoside - soaked nitrocellulose filters that were then screened by overlay assays ( mcpherson et al ., 1994 ) using a gst fusion protein encoding the sh3 domain of endophilin i ( micheva et al ., 1997a ). positive plaques were purified by two additional rounds of screening and the cdnas were isolated and identified by dna sequence analysis . the longest clone encoding rglk was completely sequenced on both strands . polyclonal antibodies against amphiphysin i / ii ( ramjaun et al ., 1997 ) and endophilin i ( 1903 ) ( micheva et al ., 1997a ) were previously described . monoclonal anti - flag m2 and anti - phospho - jnk ( thr183 / tyr185 ) antibodies were purchased from sigma and new england biolabs , respectively . a polyclonal anti - rglk antiserum was raised in rabbits against a gst fusion protein encoding amino acids 276 to 541 of rglk ( gst - glk 276 - 541 ), and anti - rglk antibodies were affinity - purified from the serum as described ( sharp et al ., 1993 ) using a his6 - tagged version of the fusion protein . a gst fusion protein encoding the sh3 domain of endophilin i was previously described ( micheva et al ., 1997a ) gst fusion protein constructs of glk , encoding various regions of the regulatory domain , were generated using an rglk cdna clone as a template in pcr reactions . the reactions were performed with the forward primer 5 ′- gcgggatcccctctgacgaggtctttg ( nucleotides 826 - 843 ) and the following reverse primers : - gst - glk 276 - 541 , 5 ′- gcgcccgggtcaggcacagtggatttt caag ( nucleotides 1605 - 1623 ); - gst - glk 276 - 498 , 5 ′- gcgcccgggtcagttcgtgcctctct gctc ( nucleotides 1477 - 1494 ); - gst - glk 276 - 445 , 5 ′- gcgcccgggtcaccctgatgaggga catc ( nucleotides 1319 - 1335 ); - gst - glk 276 - 406 , 5 ′- gcgcccgggtcacaaggttgaatgttta gagtc ( nucleotides 1198 - 1218 ). the resulting pcr products were subcloned into the corresponding bamhi and smai sites of pgex - 2t ( pharmacia biotech inc .) and the resulting gst fusion proteins were expressed and purified using standard procedures . mammalian expression constructs for full - length endophilin i and endophilin i lacking the sh3 domain ( delta sh3 ) were generated by digesting the corresponding gst fusion protein constructs ( micheva et al ., 1997a ) with bamhi and ecori , with the resulting inserts subcloned into the corresponding sites of pcdna3 ( invitrogen ). an n - terminal green fluorescent protein ( gfp ) tagged mammalian expression construct for the sh3 domain of endophilin i was generated by pcr using the full length cdna ( sparks et al ., 1996 ) as a template , with the forward primer 5 ′- gcgagatctctcagccaagaagggaatatc , and the reverse primer 5 ′- gcggaattctcaatggggcagagcaaccag . the resulting pcr product was digested with bgl ii and ecori , and subcloned into the corresponding sites of pegfp - c2 ( clontech ). to generate a construct encoding rglk with an n - terminal flag - tag , pcr was performed with the forward primer 5 ′- gcgaagcttgccaccatggactacaaagacgatgacgacaaacc gcaggaggacttcg ( nucleotides 34 - 49 ), which includes an initiation atg codon within the context of a kozak consensus sequence ( kozak , 1991 ) and a flag epitope ( dykddddk ), and the reverse primer 5 ′- gcgggatcctcactgtgtgacaaagggatg ( nucleotides 808 - 825 ) the resulting pcr product was digested with hindiii and kpni and subcloned into the same sites at the 5 ′- end of the longest rglk library clone in pbluescript . the resulting flag - tagged rglk construct was then digested with hindiii and bamhi sites and the insert was subcloned into the same sites in pcdna3 . flag - tagged gck ( yuasa et al ., 1998 ) and jnk constructs were generous gifts from dr . john kyriakis ( massachusetts general hospital ), and from dr . nathalie lamarche - vane ( mcgill university ), respectively . post - nuclear supernatants ( pns ) of various adult rat tissues were prepared in buffer a ( 20 mm hepes - oh , ph 7 . 4 , 0 . 83 mm benzamidine , 0 . 23 mm phenylmethylsulfonylfluoride , 0 . 5 μg / ml aprotinin , and 0 . 5 μg / ml leupeptin ) as described ( ramjaun et al ., 1997 ). for gst fusion protein binding assays , pns from adult rat brain was centrifuged at 45 , 000 rpm in a sorval t - 865 rotor for 1 hour at 4 ° c . and the soluble supernatant was diluted to 2 mg / ml in buffer a . triton x - 100 was added to 1 % final and 1 ml aliquots were incubated o / n at 4 ° c . with approximately 25 μg of gst fusion proteins pre - bound to glutathione - sepharose . the beads were subsequently washed three times in 1 ml of buffer a with 1 % triton x - 100 . eluted with sds - page sample buffer , and prepared for western blot analysis . for binding assays from cultured cells , 10 cm 2 dishes of transfected hek - 293t cells were washed in phosphate - buffered saline ( pbs ) ( 20 mm nah 2 po 4 , 0 . 9 % nacl , ph 7 . 4 ) and scraped into buffer b ( buffer a with 150 mm nacl ). the cells were sonicated and passed through a 25⅝ gauge needle , triton x - 100 was added to a final concentration of 1 %, and following incubation for 20 minutes at 4 ° c ., the samples were centrifuged at 75 , 000 rpm in a beckman tla 100 . 1 rotor to remove the insoluble material . aliquots of the soluble supernatant were diluted to 2 mg / ml in buffer b with 1 % triton x - 100 and 1 ml samples were incubated o / n at 4 ° c . with approximately 25 μg of gst fusion proteins pre - bound to glutathione - sepharose . the bead samples were subsequently washed in buffer b with 1 % triton x - 100 and prepared for sds - page as described above . overlay assays were performed as described ( mcpherson et al ., 1994 ). extracts from hek - 293t cells , co - transfected with flag - rglk and either endophilin i full length or endophilin i delta sh3 , were prepared as described above and were pre - cleared with either protein a - sepharose or protein g - agarose beads . the pre - cleared supernatants were then incubated with pre - immune serum or anti - endophilin i antibody ( 1903 ) coupled to protein a - sepharose beads , or with anti - flag antibody coupled to protein g - agarose beads . after 5 hr at 4 ° c ., the beads were washed extensively in buffer b with 1 % triton x - 100 and prepared for western blotting analysis . for immunoprecipitations from tissue , rat brains were homogenized in buffer a containing 0 . 3 m sucrose using a glass - teflon homogenizer with 9 strokes at 900 rpm . the homogenate was centrifuged at 800 × g max for 5 minutes and the supernatant was then centrifuged at 16 , 000 × g max for 15 minutes . nacl was added to 150 mm and the sample was incubated for 15 minutes at 4 ° c . before being centrifuged at 45 , 000 rpm in a sorval t - 865 rotor for 1 hour at 4 ° c . the resulting soluble material was made to 1 % in triton x - 100 and was pre - cleared by incubation with protein a - sepharose . the pre - cleared supernatants were then incubated with pre - immune serum or anti - rglk antibody ( 2467 ) coupled to protein a - sepharose beads . after 5 hr at 4 ° c ., the beads were washed extensively in buffer b with 1 % triton x - 100 and prepared for western blotting analysis . immunofluorescence analysis of frozen brain sections and hippocampal neurons in culture rats were anesthetized and perfused through the ascending aorta with 200 ml of 0 . 1m sodium phosphate monobasic , ph 7 . 4 ( phosphate buffer ; pb ) followed by 400 ml of 3 . 5 % paraformaldehyde in pb and 200 ml of 10 % sucrose in pb . the brains were dissected out , cryoprotected in 30 % sucrose , and 30 μm sections were prepared on a freezing microtome . the sections were then washed in 0 . 1m tris , ph 7 . 4 ( tris buffer ), permeabilized in 0 . 3 % triton x - 100 for 10 minutes , blocked in 5 % bsa and 5 % ngs for 30 minutes , and incubated with primary antibody in tris buffer containing 1 % bsa for 2 to 3 days at 4 ° c . the sections were then washed in 0 . 1m trs buffer with 1 % bsa and 0 . 1 % triton x - 100 , incubated with fluorescent secondary antibody at room temperature for 2 hours in the same buffer , washed , mounted , and dehydrated before observation . dissociated cell cultures were prepared from the ca3 region of hippocampi from p1 rat pups as described ( hussain et al ., 1999 ). hek 293t cells were co - transfected with flag - tagged jnk and a variety of constructs as indicated in the figure legends . fourty - eight hours post transfection , the media was removed and the cells were scraped and boiled in sample buffer . the samples were separated on sds - page and used for western blot analysis identification and cloning of rat germinal center kinase - like kinase as an endophilin i - binding protein a key requirement in understanding the complete range of endophilin i functions is the identification of its full complement of sh3 domain - binding partners . we thus screened a rat brain expression library with a gst fusion protein encoding the sh3 domain of endophilin i . from a total of approximately six - hundred thousand clones screened , eighteen encoded potential endophilin i - binding proteins ( data not shown ). as expected , the majority of the clones ( eight ) encoded for synaptojanin 1 . only one clone was found to correspond to dynamin i . interestingly , three independent , non - amplified isolates were found to encode for a rat homologue of the human serine / threonine protein kinase , germinal center kinase - like kinase ( hglk ) ( diener et al ., 1997 ) the longest rat glk ( rglk ) clone isolated was sequenced on both strands to generate a coding sequence that aligned to hglk , starting at amino acid 3 of its published sequence ( diener et al , 1997 ), and appeared to be complete at the c - terminal end . to obtain the extreme 5 ′- end , we searched the database of expressed - sequence tags ( dbest ) with sequence from near the 5 ′- end of our isolated clones . four overlapping rat ests were identified leading to a complete rglk coding sequence containing eleven additional amino acids ( fig1 a ). these amino acids , which extend the rglk sequence eight amino acids beyond the predicted start of hglk , were homologous to other members of the gck family ( data not shown ). the differences in the extreme n - terminal end of hglk and rglk may represent a species difference . however , multiple human est clones were identified , which when aligned with the 5 ′- end of hglk , could define an n - terminal sequence identical to that for rglk ( data not shown ). protein alignments revealed that the n - terminal kinase domain of rglk is 99 % and 72 % identical to hglk and hgck , respectively ( fig1 b ). the c - terminal regulatory domain of rglk , which includes a proline - rich region with multiple sh3 domain consensus - binding sites is 95 % identical with hglk and 44 % identical with hgck ( fig1 b ). endophilin i interacts specifically with rglk through an sh3 domain - mediated interaction to confirm the endophilin i - rglk interaction , we transfected hek - 293t cells with a construct encoding rglk containing an n - terminal flag tag ( flag - rglk ). the expressed protein was shown to bind strongly to a gst - endophilin i sh3 domain fusion protein ( gst - sh3 ) but not to gst alone ( fig2 a , top panels ). to further demonstrate the interaction specificity , we assessed the binding of flag - tagged gck ( yuasa et al ., 1998 ) ( generously provided by dr . john kyriakis ). no binding of flag - gck to the sh3 domain of endophilin i was detected ( fig2 a , top panels ). we next performed the converse pull - down experiments . full - length endophilin i , expressed in hek - 293t cells , binds specifically to a gst fusion protein encoding the proline - rich core of rglk ( gst - glk 276 - 541 ; fig2 a , bottom panels ). in contrast , an endophilin i construct lacking the sh3 domain ( endophilin i delta sh3 ) did not bind to the same rglk fusion protein ( fig2 a , bottom panels ). to demonstrate the interaction in cells , lysates from hek - 293t cells , co - transfected with flag - rglk and full - length endophilin i , were subjected to immunoprecipitation analysis with an anti - endophilin i antibody ( 1903 ) and an anti - flag antibody . immunoprecipitation of either endophilin i or flag - rglk led to the co - immunoprecipitation of the respective binding partner ( fig2 b ). in control experiments , no co - immunoprecipitation was seen when cells were co - transfected with flag - rglk and an endophilin i construct lacking the sh3 domain ( endophilin i delta sh3 ) ( fig2 b ). the endophilin i - rglk interaction was also demonstrated from brain tissue . the gst - glk 276 - 541 fusion protein strongly affinity - selected endophilin i from brain extracts , whereas amphiphysin i and ii , two major sh3 domain - containing proteins in brain ( ramjaun et al ., 1997 ), did not interact with the fusion protein ( fig2 c ). the proline - rich core of rglk contains multiple consensus sh3 domain - binding sites ( fig3 c ). to identify the proline - rich motif ( s ) responsible for endophilin i binding , we generated gst fusion protein constructs in which we deleted increasing amounts of the c - terminus of the rglk regulatory domain ( fig3 a ). whereas a construct consisting of amino acids 276 to 498 of rglk ( gst - glk 276 - 498 ) bound strongly to endophilin i from brain extracts , a construct encoding amino acids 276 to 445 ( gst - glk 276 - 445 ) showed weak binding and a construct encoding amino acids 276 to 406 ( gst - glk 276 - 406 ) failed to bind ( fig3 b ). this indicated that the major endophilin i - binding site was located between amino acids 445 and 498 of rglk with a second , much weaker site between amino acids 405 and 446 . within the region of rglk from amino acids 445 to 498 is the sequence pprppppr ( fig3 c ), which closely conforms to the previously described sh3 domain - binding sequence preference for the endophilins ( cestra et al ., 1999 ), suggesting that it is the major endophilin i - binding site in rglk . members of the gck family have been reported to interact with a number of different sh3 domain - containing proteins ( kyriakis , 1999 ). to determine whether rglk has multiple sh3 domain binding partners , we performed overlay assays of brain extracts with the gst - rglk fusion protein constructs described above . remarkably , gst - glk 276 - 541 reacted with a single 40 kda band that perfectly co - migrated with endophilin i as determined by western blot with the endophilin i antibody 1903 ( fig4 ). gst - glk 276 - 498 , which like gst - glk 276 - 541 contains the major endophilin i - binding site , also bound to the 40 kda band , whereas the fusion proteins lacking the endophilin i - binding sequence ( gst - glk 276 - 445 , and gst - glk 276 - 406 ) did not ( fig4 ). a similar specificity was seen with overlay assays of recombinant endophilin i ( data not shown ). together , these data suggest that endophilin i is a major rglk - binding protein in brain and provide compelling evidence for the specific nature of the endophilin i - rglk interaction . an anti - rglk antibody was generated through injection of the gst - glk 276 - 541 fusion protein . following affinity purification , the antibody ( 2467 ) specifically recognized a 100 kda band in rat brain extracts ( fig5 a ), consistent with the predicted molecular mass of 98 , 689 da for rglk . to confirm that the antibody recognizes rglk , we transfected cells with flag - rglk and blotted the cell lysates with anti - flag or anti - rglk antibody 2467 . both antibodies recognized an identically sized band of ˜ 100 kda in lysates from transfected cells but not in lysates from non - transfected cells ( fig5 a ). to further ensure that the 100 kda band in brain extracts is indeed rglk , we used the sh3 domain of endophilin i in affinity chromatography experiments . gst - sh3 but not gst alone strongly interacts with the 100 kda band recognized by antibody 2467 ( fig5 b ). we next explored the tissue distribution of rglk using the affinity - purified antibody . previously reported northern blot data suggested a ubiquitous distribution for the hglk mrna ( diener et al ., 1997 ). surprisingly , rglk protein , like endophilin i ( micheva et al ., 1997a ), is strongly expressed in adult and embryonic day 18 ( e18 ) brain and is weakly expressed in testis ( fig6 ), but is only seen in a variety of non - neuronal tissues upon extensive over - exposure of the blots ( data not shown ). to demonstrate that endophilin i interacts with rglk in brain tissue , we immunoprecipitated rglk from solubilized rat brain extracts using anti - sera 2467 and blotted the resulting immunoprecipitates with rglk and endophilin i antibodies . anti - sera 2467 immunoprecipitates rglk and leads to co - immunoprecipitation of endophilin i ( fig6 ). western blot analysis of dissected brain regions revealed strong expression of rglk in the hippocampus ( data not shown ). thus , to determine if rglk was expressed in neurons , we performed immunofluorescence analysis on neuronal cultures prepared from the ca3 region of the hippocampus . interestingly , rglk was detected in neurons , predominantly in large , punctate structures , possibly representing large vesicular elements ( fig7 a ). the puncta were concentrated in the peri - nuclear region but they also extended into dendrites and their growth cones ( fig7 b ). the enrichment in growth cones , which was only detected in a fraction of the neurons , was due to an increase in the density of rglk positive puncta in the growth cone area ( fig7 b ) as well as to an increase in the content of rglk per puncta ( fig7 c ). we next examined the regional distribution of rglk in adult rat brain . rglk was detected in specific neuronal populations in the cerebral cortex , the cerebellum , and the mid brain ( data not shown ). in the hippocampus , rglk was strongly expressed in the ca3 and ca2 pyramidal neurons ( fig8 a , b ) and in neurons from the hillus ( fig8 a ), but was only weakly detectable in ca1 pyramidal cells ( fig8 b ) and was not detected in dentate granule cells ( fig8 a ). rglk was strongly expressed in the substantia nigra pars compacta but was absent from the substantia nigra pars reticulata ( fig8 c ). higher power images of ca3 pyramidal neurons reveals staining that is present throughout the cell body and proximal dendrites ( fig8 b inset ). it was previously reported that overexpression of hglk leads to jnk activation in transfected hek - 293 cells ( diener et al ., 1997 ). we thus used this system to determine if endophilin i functions in glk - mediated jnk activation . specifically , we monitored jnk activation using an anti - phospho jnk antibody following transfection of glk and different endophilin i constructs . consistent with diener et al ., ( 1997 ), we find that overexpression of rglk is sufficient to activate jnk ( fig9 a ). interestingly , overexpression of full - length , untagged endophilin i along with rglk leads to an increase in jnk activation versus expression of glk alone ( fig9 a ). quantitative analysis of 8 independent experiments revealed a statistically significant ( p & lt ; 0 . 05 ; two - tailed t - test ) 2 . 01 - fold stimulation . further , overexpression of a gfp - tagged form of the sh3 domain of endophilin i or of the untagged n - terminus of endophilin i lacking the sh3 domain ( delta sh3 ), completely blocked glk - mediated jnk activation ( fig9 a ). given that the sh3 domain of endophilin i was expressed with a gfp tag , we sought to further demonstrate the specificity of this construct to block rglk - mediated jnk activation . thus , jnk activation was measured following overexpression of rglk with the gfp - sh3 domain construct or with gfp alone . whereas the gfp - endophilin i sh3 domain blocked rglk - mediated jnk activation , gfp alone had no effect ( fig9 b ). endophilin i is a brain - specific protein implicated in clathrin - mediated endocytosis , in part through its sh3 domain - dependent interactions with synaptojanin 1 , dynamin i , and amphiphysin i and ii . in order to identify additional binding partners for endophilin i , we used a gst fusion protein encoding its sh3 domain to screen a rat brain cdna expression library . of the eighteen positive cdnas isolated , those encoding synaptojanin 1 were the most abundant . the second most abundant group of cdnas encoded for the rat germinal center kinase - like kinase ( rglk ). it was particularly striking that more independent clones of rglk were isolated than for the abundant brain protein and established endophilin i - binding partner , dynamin i . the specificity of the interaction of endophilin i with rglk was further demonstrated using in vitro binding assays . whereas the sh3 domain of endophilin i bound strongly to rglk , it did not bind to the rglk homologue gck , and conversely , the proline - rich domain of rglk bound to endophilin i but not to the abundant sh3 domain - containing proteins , amphiphysin i and ii . in fact , overlay assays of brain extracts with the rglk proline - rich domain detected endophilin i exclusively , suggesting that endophilin i is a major rglk - binding protein in brain . previously , we performed overlays with the sh3 domain of endophilin i that revealed dynamin i and synaptojanin 1 as the major endophilin i - binding partners in brain ( micheva et al ., 1997a ). however , dynamin i and rglk co - migrate on sds - page at 100 kda so it is possible that the 100 kda band detected in these experiments represented a mixture of dynamin i and rglk . together , these data suggest a strong and highly specific interaction of endophilin i with rglk . the potential significance of the endophilin i / rglk interaction is highlighted by its occurrence in vivo . specifically , endophilin i and rglk can be co - immunoprecipitated following co - transfection in mammalian cells as well as from brain extracts . to further confirm the functional relevance of the interaction , we investigated whether endophilin i expression regulates jnk activation . overexpression of hglk in mammalian cells is sufficient to activate jnk ( diener et al ., 1997 ) interestingly , we find that overexpression of full - length endophilin i increases rglk - mediated jnk activation in the same system . further , overexpression of the isolated n - terminus or sh3 domain of endophilin i blocks the activation of jnk stimulated by rglk . the mechanism by which endophilin i functions in rglk - mediated jnk activation is unknown . however , for hglk , deletion of the regulatory domain , including the sh3 domain - binding sites , results in a kinase that has full catalytic activity but which is significantly impaired in its ability to activate jnk ( diener et al ., 1997 ). this data is consistent with a model in which the regulatory domain targets rglk for interactions necessary for jnk activation . thus , it is interesting to speculate that endophilin i may function as an adaptor protein to target rglk to an upstream activator or to a downstream effector functioning in jnk activation . in fact , other gcks appear to undergo specific targeting events . for example , the group 1 gck family member hpk1 is activated following its recruitment to the egf receptor via interactions with the sh3 domain - containing adaptor protein grb2 ( anafi et al ., 1997 ). gck itself is targeted to the golgi complex via its interactions with the membrane trafficking protein rab8 ( ren et al ., 1996 ) irrespective of the precise mechanism , these data suggest a functional role for endophilin i in glk activation of jnk . previous studies have established a role for endophilin i in clathrin - mediated endocytosis ( simpson et al ., 1999 ; ringstad et al ., 1999 ; gad et al ., 2000 ). our data , demonstrating a functional role for endophilin i in rglk - mediated jnk activation , suggest an additional , and perhaps complementary role for endophilin i in signaling via the jnk pathway . a link between endocytosis and cell signaling was originally suggested by the observation that active egf receptor signaling complexes undergo endocytosis and continue to signal on endosomes ( di guglielmo et al ., 1994 ). it was then demonstrated that endocytosis is necessary for full activation of erk½ map kinases following egf stimulation ( vieira et al ., 1996 ). in fact , recent studies have suggested that a broad range of receptors require endocytosis for erk½ activation including tyrosine - kinase receptors and g - protein - coupled receptors ( ceresa and schmid , 2000 ). endocytosis may be necessary to allow for access of receptors or their downstream signalling components to intracellular pools of erk½ ( kranenburg et al ., 1999 ). endocytosis may also function in the compartmentalization of signaling complexes , allowing for an additional level of specificity in signaling pathways ( defea et al ., 1999 ; zhang et al ., 2000 ). thus , it is an attractive hypothesis that endophilin i is a component of a pathway providing a novel link between endocytosis and jnk signaling . however , direct evidence to this effect is lacking and the described ability of endophilin i to function in jnk activation may occur independent of its role in endocytosis . previous northern blot analysis demonstrated that hglk has a ubiquitous tissue distribution ( diener et al , 1997 ). to further characterize the properties of rglk , we generated a polyclonal antibody against the protein . extensive analysis of the affinity - purified antibody revealed that it is specific for rglk . western blot analysis with this antibody revealed that rglk protein is expressed in multiple tissues but that its expression is greatly enriched in brain . to determine if rglk was expressed in neurons , we performed immunofluorescence analysis of rglk in hippocampal neurons maintained in culture for two days . the neurons displayed strong rglk staining that was found in puncta that were concentrated in the peri - nuclear region but which extended into neurites including nerve - terminal growth cones . previously , ringstad et al ( 1997 ) examined the distribution of endophilin i in hippocampal neurons maintained in culture for two weeks and determined that the protein was concentrated in nerve terminals but also displayed a diffuse cytoplasmic staining . thus , complexes of endophilin i with rglk may occur in the rglk - positive vesicular compartment . the identification of the rglk - positive vesicles is currently under investigation . the neuronal enrichment of rglk distinguishes it from most other group i gcks , which are ubiquitously distributed or are expressed predominantly in lymphoid tissue ( katz et al ., 1994 ; hu et al ., 1996 ; kiefer et al ., 1996 : diener et al ., 1997 ; shi and kehrl , 1997 ; fu et al ., 1999 ; yao et al ., 1999 ; moore et al ., 2000 ), but is similar to the recently described gck family member mink ( dan et al ., 2000 ). thus , rglk may play a prominent role in jnk activation in neurons . it was therefore intriguing to observe that in brain , rglk is expressed in neuronal populations that undergo jnk - mediated neuronal cell death and degeneration ( mielke and herdegen , 2000 ). for example , within the hippocampus , the ca3 pyramidal cells strongly express rglk whereas ca1 pyramidal cells and granule cells of the dentate gyrus show limited or no expression . endophilin i is also strongly expressed in the cell bodies of ca3 neurons but is detected at much lower levels in the cytoplasm of ca1 neurons ( micheva et al ., 1997a ). it has long been appreciated that ca3 neurons are prone to excitotoxic cell death ( nadler et al ., 1980 ), a process mediated through activation of the jnk pathway ( yang et al , 1997 ). another area with high rglk expression is the substantia nigra pars compacta that undergoes jnk - mediated apoptotic cell death following axonal damage ( heregen et al , 1998 ; oo et al ., 1999 ) or treatment of nigral neurons in vitro with the dopaminergic neuron toxin mptp ( saporito et al ., 1999 ). although correlative , these results may suggest a role for rglk in neuronal cell death . another situation in which neurons undergo jnk - mediated apoptotic cell death is in huntington &# 39 ; s disease ( hd ). hd is caused by an expansion of a cag repeat ( encoding polyglutamine ) in the huntingtin protein of hd patients ( the huntington &# 39 ; s disease collaborative research group , 1993 ). expression of polyglutamine - expanded huntingtin in a hippocampal cell line induces apoptosis via jnk activation ( liu , 1998 ; liu et al ., 2000 ), suggesting a role for jnk activation in the pathophysiology of hd . adjacent to the polyglutamine region is an sh3 domain - consensus binding site that interacts with endophilin iii with greatly increased affinity when huntingtin contains a glutamine expansion in the pathological range ( sittler et al ., 1998 ). endophilin iii is highly related to endophilin i ( sparks et al ., 1996 ; giachino et al ., 1997 ; ringstad et al ., 1997 ; so et al ., 1997 ) and in fact , the sh3 domain of endophilin iii binds rglk in vitro ( data not shown ). thus , it is intriguing to speculate that in hd , polyglutamine - expanded huntingtin may compete with glk for binding to endophilin i and / or iii leading to a disregulation of jnk signaling via disruption of endophilin / glk interactions . studies are underway to determine if there is a direct role for endophilin / glk interactions in hd . the invention provides methods for detecting agents such as drugs that can alter the ability of members of the endophilin protein family to associate with the germinal center kinase - like kinase ( glk ) protein , and methods for detecting agents that induce dissociation of a bound complex formed by the association of an endophilin family member and the glk protein . an example of a screening assay for detecting such agents is provided in fig1 and is described in the example 1 below . as used herein , the term “ agent ” means a chemical compound that can be useful as a drug . the screening assay described herein is particularly useful in that it can be automated , which allows for high through - put screening of randomly designed agents to identify useful drugs which can alter the ability of the endophilins and glk to associate . for example , a drug can alter the ability of the endophilin member to associate with glk by decreasing or inhibiting the binding affinity of the endophilin with glk . such a drug could be useful where it is desirable to increase the concentration of unbound glk in a cell , and therefore modulate the glk - mediated jnk pathway which may have effects on apoptosis , alternatively , a drug can be useful for increasing the affinity of binding of endophilin with glk , that may be desirable for inducing the opposing regulatory effects on the glk - mediated jnk pathway and apoptosis . the drug screening assay can utilize an endophilin family member or , as exemplified in fig1 , an endophilin fusion protein such as the endophilin sh3 domain glutathione - s - transferase ( gst ). the endophilin or endophilin fusion protein is characterized , in part , by having an affinity for a solid substrate as well as having an affinity for glk . for example , when endophilin is used in the assay , the solid substrate can contain a covalently - attached anti - endophilin antibody . alternatively , if an endophilin - gst fusion protein is used in the assay , the solid substrate can contain covalently - attached glutathione , which is bound by the gst component of the endophilin - gst fusion protein . the drug screening assay can be performed by allowing the endophilin or endophilin - fusion protein to bind to the solid support , then adding glk , together with a drug to be tested ( see example 1 , below ). control reactions will not contain the drug . following incubation of the reaction mixture under conditions known to be favorable for the association , for example of the endophilin and glk in the absence of the drug , the amount of glk specifically bound to the endophilin in the presence of the drug can be determined . for ease of detection of binding , the glk protein can be labeled with a detectable moiety , such as a radionuclide or a fluorescent label ( see example 1 , below ). by comparing the amount of specific binding of the endophilin and glk in the presence of a drug as compared to the control level of binding , a drug that increases or decreases the binding of the endophilin with glk can be identified . thus the drug screening assay provides a rapid and simple method for selecting drugs having a desirable effect on the association of the endophilins with glk . this example describes an assay useful for screening for agents such as drugs that alter the affinity of binding of an endophilin family member and the glk protein . fig1 presents a scheme for using an endophilin family member in a drug screening assay that is suitable for automated high through - put random drug screening . a cdna encoding the mouse sh3 domain of endophilin i is subcloned into the pgex - 2t prokaryotic expression plasmid ( pharmacia ; piscataway , n . j .) to produce glutathione - s - transferase ( gst ) endophilin i sh3 domain fusion protein in e . coli . gst - endophilin i sh3 domain fusion protein is affinity purified using glutathione - sepharose ( sigma chem . go . ; st . louis , mo .). following loading of the gst - endophilin i sh3 domain , the column is washed with pbs ( ph 7 . 4 ), containing 1 % tx - 100 , to remove irrelevant proteins . the specific recombinant fusion protein is eluted using excess glutathione in pbs ( ph 7 . 4 ). following dialysis , the gst - endophilin i sh3 domain fusion protein is immobilized to solid supports taking advantage of the ability of the gst fusion protein to specifically bind glutathione . the assay can utilize any form of glk that includes the proline - rich endophilin - binding site . a cdna encoding the proline - rich region of glk is subcloned into the baculovirus transfer vector , pacsg - his , which produces histidine - tagged fusion proteins in sf9 cells ( pharmingen , inc .). the recombinant protein is affinity - purified by standard methods using nickel - chelation chromatography , essentially as described by smith and johnson , gene 67 : 31 - 40 ( 1988 ), which is incorporated herein by reference . the recombinant glk fusion protein can be eluted in imidazole ( ph 6 . 0 ). following dialysis , the his - glk fusion protein can be chemically modified to permit easy detection . several different chemical modifications can be used to attach a detectable moiety such as a fluorescent molecule , a radiolabel or another protein which can be detected using a specific antibody or other specific reagent . for example , fluorescein - 5 maleimide can be attached as a fluorescent tag to the glk protein . various agents such as drugs are screened for the ability to alter the association of endophilin i and glk . the agent , endophilin i and fluorescent - glk are added together , incubated for 30 min to allow binding , then washed to remove unbound fluorescent - glk protein . the relative amount of binding of fluorescent - glk protein in the absence as compared to the presence of the agent being screened is determined by detecting the relative light emission of the fluorochrome . the assay is readily adaptable for examining the interaction of other endophilin family members with glk , such as endophilin ii and iii . the screening assay is useful for detecting agents that alter the association of other endophilin family members and the glk protein by increasing or decreasing their binding affinity . as shown in fig1 , glioma cell lines ( a ) u343 and ( b ) 373 were plated in 96 - well plates , infected with nothing , control gfp adenovirus or glk adenovirus and analyzed for survival by mtt dye incorporation . glioma cell lines u343 and u373 ( p53 null ) were plated at 5000 cells / well on 96 - well plates and infected 24 hours later with increasing mois ( 0 , 50 , 100 , 250 and 500 ) of recombinant gfp or glk adenovirus . appropriate titers of virus were diluted into 10 % of the culture volume containing deae - dextran , incubated for 30 min at 25 ° c . and then added directly to the cells . twenty - four hours post - infection , cells were assayed for viability using 3 ( 4 , 5 - dimethylthiozol - 2 - yl ) 2 , 5 - diphenyltetrazolium bromide ( mtt ; sigma ), which was added at a final concentration of 1 mg / ml for 4 hours . the reaction was ended by the addition of 1 volume of solubilization buffer ( 20 % sds , 10 % dimethylformamide , and 20 % acetic acid ). after overnight solubilization , specific and non - specific absorbance were read at 550 and 690 nm , respectively , and the average of 6 wells per condition were compared . although the present invention has been described hereinabove by way of preferred embodiments thereof , it can be modified , without departing from the spirit and nature of the subject invention as defined in the appended claims . anafi , m ., kiefer , f ., gish , g . d ., mbamalu , g ., iscove , n . n ., and pawson , t . 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