Patent Application: US-201013500902-A

Abstract:
the present invention relates to human serine / threonine kinase ulk3 and its ability to regulate gli transcription factors ; mediators of shh signaling . this disclosure demonstrates that ulk3 enhances endogenous and over - expressed gli1 and gli2 transcriptional activity in cultured cells , and ulk3 alters subcellular localization of gli1 . according to this disclosure ulk3 is an autophosphorylated kinase and phosphorylates gli proteins in vitro . a peptide sequence in gli1 c - terminus that is phosphorylated by ulk3 is provided in this disclosure . ulk3 catalytical activity is shown to be crucial for its function in shh pathway . this disclosure shows that serine / threonine kinase ulk3 is involved in the shh pathway as a positive regulator of gli proteins . furthermore , a therapeutic method in shh dependent human disorders is disclosed by pharmacological inhibition of ulk3 kinase activity . identification of ulk3 substrate sequence in gli1 allows the design of peptide - based modulators of its kinase activity .

Description:
abbreviations used in this disclosure : ci , cubitus interruptus ; ck1 , casein kinase 1 ; fcs , fetal - calf serum ; fu , fused ; gsk3 , glycogen synthase kinase 3 ; ip , immunoprecipitation ; kb , kinase buffer ; ne , nuclear extract ; pka , protein kinase a ; pei , polyethylenimine ; pic , protease inhibitor cocktail ; shh , sonic hedgehog ; stk36 , serine / threonine kinase 36 ; qrt - pcr , quantitative real - time pcr ; swm , stain wash medium ; ulk , unc - 51 - like kinase ; wce , whole cell extract . in this disclosure we show the cloning of human serine / threonine kinase ulk3 that has been annotated as belonging to unc - 51 - like family of serine / threonine kinases , but shares similarity with stk36 and dfu proteins . we show that ulk3 is an autophosphorylated kinase . in cultured cells ulk3 is able to enhance endogenous and overexpressed gli1 and gli2 transcriptional activity and to induce nuclear translocation of gli1 . we show that ulk3 phosphorylates gli proteins in vitro , and gli1 has at least two phosphorylation sites situated in n - and c - terminus of the protein . we have found that , in contrast to stk36 , the kinase - deficient mutants of ulk3 are inactive indicating that functional kinase domain of ulk3 is required for the regulation of gli protein activity . also we show that ulk3 expression is higher in fetal brain and in a number of postnatal tissues where shh signaling is known to be active . our data suggests that ulk3 is involved in shh pathway as a positive regulator of gli proteins . in this disclosure we identify human ulk3 as a serine / threonine kinase regulating positively shh pathway in mammalian cells . it has been shown previously that protein kinase dfu is absolutely required for the activation of hh pathway in drosophila . dfu works in a concert with cos2 , dsmo and dsufu , forming a microtubule - binding complex that controls activity of ci transcription factor [ 18 - 20 , 22 , 23 ]. in spite of overall hh pathway conservation between vertebrates and invertebrates , the function of those proteins is not fully conserved in mammals . in drosophila cos2 plays a central role in regulating the activity of ci [ 40 , 41 ], but mammalian orthologues of cos2 , kif7 and kif27 , have no effect on shh signaling [ 4 ]. the cytoplasmic c - terminus of dsmo , that is extremely important for hh signaling in drosophila , is not conserved in mammalian smo , and , moreover , mouse smo c - terminus is not required for shh signal transduction [ 4 , 22 ]. in drosophila sufu gene function is dispensable for hh signaling and sufu protein has only a slight negative effect on ci , whereas mouse sufu −/− mutants are not viable and inhibiting effect of mammalian sufu on gli proteins is very influential [ 42 , 43 ]. taking into account the divergences of hh signaling in drosophila and mammals on molecular level , it is not surprising that the role of mammalian fu in hh pathway also differs from that in drosophila . mammalian orthologue of dfu , stk36 , was identified based on sequence homology [ 26 ]. while stk36 is able to positively regulate gli proteins in cultured cells , it is dispensible for shh signaling during mouse development [ 30 , 31 ]. accordingly , additional kinases may be involved in the mammalian hh pathway . this disclosure shows that ulk3 participates in gli regulation . previously , ulk3 has been annotated as belonging to the family of unc - 51 - like kinases that comprises also ulk1 , ulk2 and ulk4 proteins . this disclosure provides however , that ulk3 diverges from other members of the family . the bioinformatic analysis suggests that ulk3 is the closest homologue of stk36 in humans ( fig1 a ). we cloned human ulk3 full - length cdna and made two kinase inactive forms of ulk3 by introducing point mutations into the catalytic domain of the protein ( fig1 b ). analysis of ulk3 expression pattern reveals that ulk3 is widely expressed and its expression is higher in a number of tissues where shh signaling is known to be active , such as postnatal brain , hippocampus , olfactory bulb , cerebellum , optic nerve , liver and fetal brain ( fig1 c and 1d ). in the same parts of brain expression level of gli1 and ptch1 mrna was also elevated ( data not shown ). the role of shh signaling in brain development is very extensive ( reviewed in [ 44 ]). there is no direct evidence for shh role in liver development , and in healthy adult liver shh activity is low [ 45 ]. but it has been shown that cells , involved in adult liver repair , are capable of producing and responding to hh ligands [ 46 ]. in central nervous system shh signaling has been shown to contribute to neurogenesis that is going on in adult brain . hippocampus is known as one of the zones where neurogenesis occurs , and shh signaling has been shown to be implicated in control of stem cells behavior in adult hippocampus [ 47 ]. in postnatal cerebellum shh , expressed purkinje cells , regulates proliferation of the granule cells — the process required for cerebellar foliation ( reviewed in [ 44 ]). also shh signaling has been shown to be active in rodent postnatal optic nerve [ 48 ]. the role of shh signaling in olfactory bulb has not been documented yet . however , it has been shown that stem cells of subventricular zone of forebrain proliferate under the control of shh [ 49 ]. those cells migrate into the olfactory bulb , where they differentiate into functional interneurons [ 44 ]. the instant disclosure shows that shh - responsive tissues express higher levels of ulk3 mrna and accordingly that provides indirect evidence that ulk3 is involved in shh pathway in vivo . this disclosure shows that ulk3 ( seq id no : 1 ) possesses kinase activity and is able to phosphorylate itself in vitro ( fig3 a ). it has been previously shown that ulk1 and ulk2 are , also autophosphorylated kinases in vitro [ 38 , 39 ]. mouse ulk1 and ulk2 are autophosphorylated in the conserved central proline / serine rich domain of the protein . however , ulk3 lacks such domain . instead , according to bioinformatic analysis , ulk3 protein harbors a central domain contained within microtubule interacting and trafficking molecules ( mit ). besides that , the sequence mapped in dfu as sufu - interacting domain ( residues 306 - 436 , [ 20 ]) is conserved to a certain extent in ulk3 . however , c - terminal sequences responsible in dfu for interacting with cos2 and carboxyl terminus of smo are absent in ulk3 . this disclosure also provides that ulk3 is able to phosphorylate all three mammalian gli proteins in vitro ( fig3 b ). although a number of serine / threonine kinases has been proposed to be involved in modulating hh pathway both in drosophila and in vertebrates , only pka has been shown to phosphorylate directly all gli proteins , and ck1 and gsk3 have been shown to phosphorylate gli2 and gli3 following primary phosphorylation by pka [ 36 , 50 - 53 ]. pka phosphorylates gli1 in residues thr - 374 and ser - 640 [ 36 ]. gli2 and gli3 are phosphorylated by pka , ck1 , and gsk3 in a c - terminal cluster between amino acids 784 - 855 that corresponds to gli1 residues 590 - 658 [ 51 , 53 ]. according to this disclosure ulk3 phosphorylates gui ( seq id no : 2 ) in both n - terminus ( residues 1 - 426 ) and c - terminus ( residues 754 - 1126 ), but fragment of gli1 between residues 426 - 754 is not phosphorylated by ulk3 ( fig3 c ). phosphorylation by pka , ck1 and gsk3 has been described to elicit mainly negative effects on gli proteins ( however , see [ 14 ]). our analysis of shh pathway activation in cell culture identifies ulk3 as a positive regulator of the pathway . cotransfection of ulk3 together with gli1 or gli2 in shh - l2 cells shows that ulk3 is able to potentiate the transcriptional activator function of both of them ( fig2 a ). also , we have found that ulk3 , comparing with its closest homologues ulk1 and stk36 , has the strongest effect on gli - dependent luciferase reporter activity . it has been previously reported that stk36 is a positive regulator of shh pathway that acts independently on its functional kinase domain . stk36 is able to induce nuclear translocation of gli1 [ 26 ]. besides that , stk36 enhances gill transcriptional activity in nih3t3c2 and sw480 cells [ 29 ] and gli2 transcriptional activity in c3h / 10t and hek293 cells [ 26 , 27 ]. here , we show that stk36 fails to induce gli1 but induces gli2 transcriptional activity in shh - l2 cells . however , stk36 possesses significantly weaker coactivator potential than ulk3 , probably due to lack of kinase activity . we show that kinase activity of ulk3 is required for activation of shh pathway in cell culture ( fig2 b ) and to induce nuclear accumulation of gli1 ( fig4 ). together with the data showing that ulk3 is able to phosphorylate all gli proteins in vitro , we suggest that ulk3 is pan - gli activating kinase in mammalian cells . the invention is now described by examples that are meant to be descriptive and by no means limiting the various embodiments of the present invention . ulk3 cdna was amplified using primers pair sense 5 ′- aatggcggggcccggctg - 3 ′ ( seq id no : 3 ) and anti - sense 5 ′- tctgctccagatggctcaca - 3 ′ ( seq id no : 4 ) from human testis cdna sample using expand long template pcr system kit ( roche applied science , bazel , switzerland ) according to manufacturer &# 39 ; s instructions . obtained pcr product was purified from agarose gel using qiaquick gel extraction kit ( qiagen , valencia calif ., usa ) and cloned to ptz57r / t vector using instaclone ™ pcr cloning kit ( fermentas , vilnius , lithuania ). ulk3 cdna ( seq id no : 5 ) was verified by sequencing and subcloned to mammalian expression vectors . ulk3pcdna3 . 1 construct was generated by cloning of ulk3 cdna into kpni and bamhi linearized pcdna3 . 1 vector ( invitrogen , carlsbad calif ., usa ). ulk3flag construct was produced by cloning of ulk3 cdna into ecori and hindui sites of pflag - cmv - 4 vector ( sigma - aldrich ). ulk3 ( k44r ) ( seq id no : 6 ) with lysine residue at position 44 mutated to arginine and ulk3 ( k139r ) ( seq id no : 7 ) harboring the same mutation at position 139 were generated from ulk3flag construct by quickchange site directed pcr mutagenesis procedure ( stratagene , la jolla calif ., usa ) using expand long template pcr system kit ( roche applied science ) and oligos carrying the appropriate point mutations . the obtained constructs were verified by dna sequencing . n - terminally tagged gli1gfp and gli1flag constructs have been described in [ 32 ]. gli2flag and gli3flag constructs were constructed in tallinn university of technology . gli2flag was generated by subcloning gli2 cdna from gli2pcdna3 described in [ 33 ] into hindiii and xbai sites of pflag - cmv - 4 vector . gli3pcdna3 . 1 construct ( described in [ 34 ]) was used for generation of gli3flag by subcloning of gli3 cdna to pflag - cmv - 4 vector . stk36pcdna3 . 1 has been described in [ 27 ]. cdna panels of 20 human tissues and 10 human brain parts were used in this experiment . levels of ulk3 mrna and mrna of housekeeping gene hprt used for normalization were detected in triplicates by quantitative real - time pcr using qpcr core kit for sybr green ( eurogentec , oslo , norway ) with lightcycler 2 . 0 ( roche applied science ) according to the manufactures &# 39 ; instructions . data was analyzed with lightcycler 4 . 05 software ( roche applied science ). the following primers were used for the assay : hek293 ( human embryonic kidney - 293 ) cells were propagated in minimum essential medium ( mem ) ( gibco , invitrogen ). nih3t3 ( mouse embryonic fibroblasts ) and their clone shh - light2 cells [ 35 ] were grown in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( d - mem ) contained 4 . 5 g / l glucose ( gibco ). cos1 cell line ( primate kidney fibroblasts ) was grown in dmem contained 1 g / l glucose ( gibco ). all growth media were supplemented with 10 % fbs ( paa ) and 100 μg / ml of penicillin / streptomycin ( invitrogen ), and shh - l2 cells growth medium was additionally supplemented with 400 μg / ml of g 418 ( sigma - aldrich - aldrich ) and 100 μg / ml of zeocine ( invitrogen ). the cells were grown at 37 ° c . and 5 % co 2 . approximately 24 h prior to transfection the cells were plated to appropriate growth dishes . cells were transfected by the expression constructs or respective empty vectors using polyethylenimine transfection agent ( pei ) ( inbio ) and as described in [ 28 ]. we used 0 . 25 μg of cdna per 1 cm 2 of plate surface area for transfections . after 3 h of transfection hek293 , nih3t3 and cos1 cells were propagated in the normal growth medium for 48 h . prior the further analysis the cells were washed twice with pbs . post - transfectional shh - l2 cells were grown in the normal growth medium for 24 h and for additional 24 h in the light medium containing 0 . 25 % fbs , washed once with pbs , lysed in passive lysis buffer ( promega , madison wis ., usa ) ( 70 ul per well of 24 - well plate format ) and subjected to luciferase assay . luciferase assay was performed as previously described [ 28 , 36 ]. briefly , firefly luciferase activity of 12 μl of the shh - l2 cells lysate was measured using luciferase assay kit ( promega ) and galactosidase activity was quantified using galacto - lightplus kit ( tropix , bedford mass ., usa ) according to the manufacturer &# 39 ; s instructions . chemiluminicence was measured using ascent fl fluoroscan ( thermo electron corporation , waltham mass ., usa ) according to the manufacturer &# 39 ; s instructions . three overlapping human gli1 domains corresponding to amino acids 1 - 433 , 426 - 754 and 726 - 1106 were cloned into pet - 15b vector ( novagen ) between ndei and bamhi sites . gli1 amino acid sequence is provided as seq id no : 2 ). the fragments were expressed in bl21 ( de3 ) plys e . coli strain overnight at 21 ° c . using 0 . 1 mm iptg and purified using ni - cam ™ hc resin ( sigma - aldrich ) according to the manufacturer &# 39 ; s recommendations . the proteins were eluted by pbs and verified by western blot ( wb ). hek293 cells were transfected with 13 . 5 μg of flag - tagged constructs expressed gli1 , gli2 , gli3 , ulk3 , ulk3 ( k44r ), ulk3 ( k139r ) or flag - cmv - 4 empty vector on 10 cm plates . the cells were lysed with 0 . 5 ml of lysis buffer ( 50 mm tris hcl , ph 7 . 4 , 150 mm nacl , 1 mm edta , 1 % triton x - 100 ) containing protease inhibitor cocktail ( pic ) ( roche applied science ). the lysates were centrifuged for 15 min at 4 ° c . and 15000 g , and the supernatants were used for immunoprecipitation ( ip ). ip was performed using anti - flag - m2 affinity gel ( sigma - aldrich ) in batch format according to the manufacture &# 39 ; s instructions . immune complexes were washed three times with 500 μl of kinase buffer ( kb ) ( 50 mm hepes , ph 7 . 4 , 20 mm mgcl 2 , 25 μm atp ) and resuspended in 12 . 5 μl of kb . one fifth of the immuno - complexes was subjected to wb and the rest was used for the kinase assay . kinase reactions were carried out in kb in a total volume of 20 μl of in the presence of 1 μci of [ γ - 32 p ] atp per reaction . we used 1 μl of the immunoprecipitated ulk3 , ulk3 ( k44r ) or ulk3 ( k139r ) mixed with 5 μl of the gli - m2 immunocomplexes or 5 μl of bacterially expressed and purified his - gli1 fragments . kinase reactions were held at 30 ° c . for 30 min and terminated by adding of 5 μl of 4 × laemmli sample buffer . proteins were resolved by sds - page . the gel was dried at 80 ° c . for 2 h , and autoradiography was performed using bio - rad personal molecular imager fx . nih3t3 cells were transfected with gli1gfp and flag - tagged expression constructs for ulk3 or ulk3 ( k139r ) in ratio 1 : 1 on 8 - chamber slides ( falcon , bd biosciences , san jose calif ., usa ). the cells were fixed with 4 % paraformaldehyde and washed three times for 5 min with stain wash medium ( swm ) ( 0 . 5 % bsa and 0 . 01 % nan 3 in pbs ). subsequent permeabilization of cells was performed at rt by 10 - min incubation in . pbs supplemented with 0 . 5 % triton x - 100 . afterwards , the cells were washed three times for 5 min with swm , blocked for 30 min in pbs containing 5 % bovine serum albumin ( bsa ), and incubated with mouse monoclonal m2 - anti - flag antibody ( diluted 1 : 1000 in swm ) for 1 h at rt with gentle agitation . after three washes of 10 min with swm , the cells were incubated for 30 min with secondary antibody alexa - fluor 568 ( invitrogen ) ( diluted 1 : 500 in swm ) mixed with nuclear stain hoechst 33342 ( diluted 1 : 100 ). cells were washed twice with swm and mounted with mowiol 4 - 88 ( sigma - aldrich ). gli1gfp protein localization was assessed in at least 200 flag - positive cells under a fluorescent microscope olympus bx61 with uplan sapo 40 × objective , and the experiment was repeated three times . confocal images were obtained with a zeiss lsm - 510 meta confocal laser - scanning microscope ( carl zeiss microimaging gmbh , germany ) equipped with plan - apochromat 63 ×/ 1 . 4 oil immersion objective . cos1 cells were transfected with flag - tagged expression constructs for gli1 combined with ulk3 , ulk3 ( k139r ) or empty vector in ratio 1 : 1 on 10 cm plates . nuclear and whole cell extracts ( ne and wce , respectively ) were prepared as described in [ 36 ]. all extracts were normalized for protein amounts measured using bsa kit ( thermo scientific ). a total 10 μg of protein were separated by sds - page and transferred to the pvdf membrane ( millipore , billerica mass ., usa ). the membrane was segmented according to the expected size of the detected proteins . obtained strips were probed with the appropriate antibodies . wb was performed using a non - blocking technique as described in [ 37 ]. mouse monoclonal m2 - anti - flag antibody ( sigma - aldrich ) was used for detection of flag - tagged gli1 , gli2 , gli3 , ulk3 , ulk3 ( k44r ) and ulk3 ( k139r ) by wb in dilution 1 : 2000 . lamin a / c was detected with mouse monoclonal anti - lamin a / c antibody ( upstate , billerica mass ., usa ) diluted 1 : 1000 . mouse monoclonal anti - his antibody ( novagen , darmstadt , germany ) diluted 1 : 1500 was used for detection of bacterially expressed and purified gli1 fragements . the secondary antibody used was hrp - conjugated goat - anti - mouse ig diluted 1 : 1000 . hek293 cells were transfected with flag - tagged wt ulk3 , ulk3 ( k139r ) or respective empty vector in two replicates . after 5 h of incubation one replicate of cells transfected with the vector was induced by shhc24ii . the cells were incubated 24 h and 72 h , washed with pbs . total rna was isolated and treated with dnasei using rnaqueous kit ( ambion ) according to manufacture &# 39 ; s instructions . cdna of 2 μg total rna was synthesised using superscript iii kit ( ambion ). levels of gli1 mrna and mrna of housekeeping gene hprt used for normalization were detected in triplicates by quantitative real - time pcr . the following gli1 primers were used for the assay : in order to find out the kinases homologous to human stk36 ( genbank accession number np056505 . 2 ), sequence of its kinase domain was subjected to comparative homology analysis against human proteins databank using blastp algorithm and blosum62 matrix ( ncbi ). our analysis revealed that ulk3 ( accession number np001092906 ) was a protein sharing the highest homology with stk36 ( 38 % of identity in the kinase domain ). using the sequence of kinase domain of dfu ( accession number np477499 . 1 ) as a query , we found that ulk3 was the second homolog of dfused protein after stk36 in human . however , the identity between ulk3 and dfused kinase domains was lower comparing to that of stk36 ( 37 % vs 51 %). on the other hand ulk3 ( seq id no : 1 ) was found to be homologous to ulk1 and ulk2 proteins that belong to unc51 subfamily ( 39 % and 37 % of identity in the kinase domain , respectively [ 38 , 39 ]). the sequences of the members of the unc51 subfamily were obtained from ncbi database ( accession numbers np — 507869 for unc51 ( c . elegance ), np648601 for atg1 ( d . melanogaster ), np003556 for ulk1 , np055498 for ulk2 , np060356 for ulk4 ). the proteins of fused and unc51 subfamilies of serine / threonine kinases were subjected to multiple sequence alignment and homology analysis using gonnet 250 matrix and clustalw program ( ebi , embl ). the homology tree was built using the calculated distances between the aligned proteins . pairwise aligment of the proteins showed that , ulk3 shared higher homology to fu subfamily of serine / threonine kinases than to unc - 51 - like kinases ( fig1 a ). taken together , the bioinformatic analysis suggests that ulk3 is a homologue of stk36 . the comparative analysis of ulk3 nucleotide sequence against human genbank was performed using ucsc genome browser . the analysis revealed ulk3 gene is situated in chromosome 15 and contains 16 exons with translation initiation codon in the first exon and stop codon in the exon 16 ( fig1 b ). ulk3 cdna was amplified by rt - pcr from adult human testis cdna using the pair of primers complementary to the predicted coding part of ulk3 . the obtained 1419nt long cdna ( seq id no : 5 ) corresponded to ulk3 mrna sequence ( genbank accession number nm001099436 . 1 ) it encodes a polypeptide of 472 amino acids ( seq id no : 1 ) with calculated molecular weight of 53 kda and contains n - terminal serine / threonine kinase domain ( amino acids 14 - 270 ). ( seq id no : 14 ) in order to produce hypothetically kinase - inactive variants of ulk3 , we mutated the highly conserved lysines in positions 44 and 139 to arginines ( ulk3 ( k44r )( seq id no : 6 ) and ulk3 ( k139r )( seq id no : 7 )). lysin residue in position 44 is situated in the atp binding pocket , and lysine in position 139 is situated in the substrate binding pocket and catalytic loop regions . the substitutions were made by site directed pcr mutagenesis procedure using the oligos carrying the appropriate mutations . ulk3 mrna is widely expressed in humans with the highest expression in fetal brain the level of ulk3 mrna expression was analyzed in 20 human tissues including fetal brain and fetal liver using quantitative real - time pcr . ulk3 mrna was detected in all tissues analyzed and the data is shown relative to the level in the heart as a tissue with the lowest level of ulk3 mrna expression ( fig1 c ). the highest expression of ulk3 was detected in fetal brain . post - natal tissues showing high level of ulk3 expression were brain , liver and kidney ; moderate amount of ulk3 mrna expression was detected in testis and adrenal gland . heart , lung , stomach , thymus , prostate and placenta showed low level of ulk3 expression . as the expression of ulk3 in the brain was higher than in most other tissues , we were interested to know if particular regions of adult human brain are responsible for high level of ulk3 expression . we analyzed 10 brain regions and found the highest level of ulk3 expression in hippocampus ( fig1 d ). the data is shown relative to the expression level in the cerebral cortex . higher levels of ulk3 mrna were detected in cerebellum , olfactory bulb and optic nerve . the lowest level of ulk3 expression was detected in spinal cord . analysis of ulk3 expression suggests that ulk3 may contribute to brain development as well as play a role in adult organism . to show that ulk3 plays a role in shh pathway we examined if ulk3 and its closest homologues ulk1 and stk36 are able to activate gli - dependent luciferase activity in shh - light2 cells ( shh - l2 ) cells . we cotransfected the constructs together with gli - expressing plasmids ( or respective empty vector ) into shh - l2 cells and assessed their effect on gli - dependent firefly luciferase activity . the obtained data was normalized with β - galactosidase values . the experiment was repeated 4 times , and results of the representative experiment are shown in fig2 a . among the kinases tested , ulk3 surprisingly demonstrated the strongest effect on gli - dependent luciferase reporter activity either alone or cotransfected with gli1 and gli2 . it was able to stimulate the luciferase activity 3 . 8 times and enhanced the transcriptional activity of over expressed gli1 and gli2 approximately 2 and 3 . 2 times , respectively . the potency of stk36 in the assay was much lower . stk36 alone failed to activate the luciferase activity and exerted no effect on gli1 ; however it enhanced gli2 activity 1 . 6 times . it is noticeable that ulk1 alone was able to activate luciferase activity 3 . 4 times ; however it had no effect on overexpressed gli1 and gli2 indicating that ulk1 may influence the shh pathway bypassing gli proteins . in order to examine if the kinase activity of ulk3 is required for the luciferase reporter activation we tested the supposed kinase - deficient variants of ulk3 , ulk3 ( k44r ) and ulk3 ( k139r ) in the same assay . in contrast to ulk3 , neither of the mutants had an effect on gli1 transcriptional activity . however , mutant ulk3 ( k44r ), if coexpressed with gli2 , demonstrated some residual activity , whereas ulk3 ( k139r ) was completely inactive ( fig2 b ). we also tested the effect of ulk3 and the mutants on the luciferase reporter when the shh pathway was activated by cotransfection with pshhn ( plasmid expressing biologically active part of shh ). ulk3 significantly affected the activated pathway inducing the luciferase activity approximately 4 . 1 times . neither of the mutants could activate the gli - luciferase reporter as efficiently as wild - type ulk3 , although ulk3 ( k44r ) had a residual positive effect on the activated pathway inducing luciferase activity 1 . 7 times . these data indicate that in contrast to stk36 and ulk1 , ulk3 is able to positively regulate the gli transcriptional activity and its kinase activity is required for that . to test if ulk3 has an autophosphorylation activity , we expressed flag - tagged wild - type ulk3 and mutants ulk3 ( k44r ) and ulk3 ( k139r ) in hek293 cells , immunopurified and subjected to in vitro kinase assay in the presence of γ - p 32 - atp . ulk3 demonstrated strong autophosphorylation activity , ulk3 ( k44r ) showed reduced efficiency in autophosphorylation , and ulk3 ( k139r ) autophosphorylation activity was almost completely absent ( fig3 a ). this data proves that ulk3 is an autophosphorylated kinase . its activity in vitro is altered by the mutations in lysines 44 and 139 — partly inhibited by mutation in lysine 44 or entirely destroyed by mutation in position 139 . we also assayed whether ulk3 is able to phosphorylate gli proteins . flag - tagged gli1 , gli2 and gli3 proteins were expressed and immunoprecipitated from hek293 cells and used as substrates for immunopurified ulk3 in the in vitro kinase assay . ulk3 was able to phosphorylate gli proteins but with different efficiency ( fig3 b ). the strongest phosphorylation signal was detected in the case of gli2 . the mutant ulk3 ( k44r ) could slightly phosphorylate gli2 ( data not shown ) which is consistent with the luciferase assay data . gli1 and gli3 were also phosphorylated by ulk3 , but with significantly lower intensity as compared to gli2 . to support these findings and identify the regions in gli1 protein that are phosphorylated by ulk3 , we used bacterially expressed his - tagged fragments of gli1 as substrate for ulk3 kinase . two of them — gli1 ( 1 - 433 ) and gli1 ( 726 - 1106 )— were phosphorylated by ulk3 . central part of gli1 ( amino acids 426 - 754 ) was not phosphorylated by ulk3 . this data shows that ulk3 phosphorylates directly gli proteins and gui has at least two phosphorylation sites situated in n - and c - terminus . ulk3 promotes nuclear localization of gli1 and kinase activity is essential to the function next we examined if overexpression of ulk3 kinase influences the subcellular localization of gli1 and gli2 proteins . as ulk3 ( k44r ) demonstrated the residual activity both in the luciferase and in vitro kinase assays and ulk ( k139r ) was completely inactive , we preferred the latter as a negative control in the immunofluorescence assay . flag - tagged ulk3 and ulk3 ( k139r ) ( or respective empty vector ) were coexpressed with gfp - tagged gli1 and gli2 in nih3t3 cells . subcellular localization of gli1 and gli2 was determined , and average values were calculated from three independent experiments . ulk3 and its mutant remained almost completely cytoplasmic . gfp - tagged gli2 localized predominantly in the nucleus and its localization was not altered by ulk3 or ulk3 ( k139r ) ( data not shown ). overexpressed gli1gfp was detected both in cytoplasm and nucleus ( fig3 a ). the distribution of gli1 was the following : 27 % of cells showed stronger signal in the nucleus , gli1 was cytoplasmic in 20 % of cells , and in 53 % of cells gli1 was distributed uniformly within the cell . expression the kinase - deficient mutant ulk3 ( k139r ) did not influence gui subcellular localization , the distribution of gli1 between nuclei and cytosol remained unchanged . under the influence of ulk3 localization of gli1 was shifted . gli1 was detected mostly in the nucleus in 70 % of cells , only in 2 % of cells retained gui in the cytoplasm , and 25 % of cells demonstrated the uniform distribution of gli1 . to confirm the data from the immunofluorescent staining , we performed cell fractionation analysis . flag - tagged gli1 and ulk3 or ulk3 ( k139r ) ( or the respective empty vector ) were coexpressed in shh - unresponsive cos1 cells , and the cells were fractionated to whole cell and nuclear extracts ( wce and ne , respectively ). the extracts were used in wb with anti - flag and anti - lamin a / c antibodies . three independent experiments were done with similar results , and the data of representative experiment are shown in fig4 b . expression of lamin a / c was analyzed as a loading control . ulk3 and ulk3 ( k139r ) were detected mostly in wces . all wces showed comparably equal expression of gli1 . analysis of nes revealed that cells cotransfected with gli1 and ulk3 retained most of the gli1 in the nuclei . cells cotransfected with gli1 and ulk3 ( k139r ) or empty vector demonstrated equal but significantly lower amounts of gli1 in the nuclear extracts . this data demonstrates that ulk3 alters the subcellular localization of gli1 but not gli2 , and the kinase activity of ulk3 is needed to induce the nuclear translocation of gli1 . wild - type ulk3 but not a kinase - deficient mutant ulk3k139r induces the expression of endogenous gm in hek293 cells next we tested if ulk3 is able to induce the endogenous gli1 expression . hek293 cells were transfected by ulk3 or respective empty vector . cells transfected with empty vector were induced by 12 nm of shhc24ii and were used as a posititve control . cells were incubated 24 h and 72 h , and total rna was isolated . level of gli1 expression was measured using qrt - pcr procedure and the obtained data was normalized by expression of housekeeping gene hprt . level of gli1 expression in cells transfected with empty vector was taken as 1 . during 72 h over expressed ulk3 was able to induce the expression of gli1 in hek293 cells ( fig5 a ). however , the kinase - deficient mutant ulk3 ( k139r ) failed to activate the expression of gui ( fig5 b ). this data proves that overexpressed ulk3 induces the endogenous expression of gui in kinase activity - dependent manner . as is shown in example 6 ulk3 phosphorylates gli1 in both n - terminus ( residues 1 - 426 ) and c - terminus ( residues 754 - 1106 ). however , the exact sites of phosphorylation have to be determined using mass spectrometry analysis . we have found the phosphorylated peptide in c - terminus of gli1 : sgsyptpspchenfvvganr ( seq id no : 15 ). this sequence corresponds to gli1 amino acid residues 961 - 981 and contains 4 potential phosphorylation sites ( 3 serine residues and 1 threonine residue ). as is evident from this disclosure , the kinase activity of ulk3 is essential factor in shh signaling . accordingly , identification of the phosphorylated peptide on c - terminus of gli1 is an essential tool in regulation of the signalling pathway . specifically , it can be used to design a high affinity inhibitor for the ulk3 kinase that is a drug candidate to enter further development . as is shown in the above examples , ulk3 is a serine / threonine kinase positively regulating the mammalian hh signaling pathway . the importance of hh signaling pathway is well known in the art and various human conditions are known to be affected by the activation / inactivation of the pathway . accordingly our novel finding provides novel means to control the pathway and provide a target for various pharmaceutical approaches . an inhibitor of the ulk3 kinase would be a desired molecule to control the hh signaling pathway . the inhibitor of protein kinase ulk3 may be designed as a competitor molecule directed to bind the kinase active site . the binding affinity of this molecule towards ulk3 may be higher or comparable to the affinities of the ulk3 substrates . the inhibitor molecule may be designed to bind the atp binding site , the protein / peptide substrate binding site , or it may be a bifunctional inhibitor binding simultaneously both of these sites . the inhibitor molecule can structurally mimic the adenosine moiety of atp , the ribose moiety or phosphate moiety of atp , or it can be any molecule exhibiting affinity and specificity to the atp binding site of ulk3 . the inhibitor competing with the protein / peptide substrate may contain a pseudosubstrate sequence based on a phosphorylation site sequence of gli , the physiological substrate of ulk3 , or some other substrate . in the case of a pseudosubstrate , the phosphoacceptor serine or threonine could be mutated to alanine or some other amino acid . as one of the phosphorylation sites in gli1 has been identified to be in seq id no : 15 in example 9 above , the inhibitor molecule could be a pseudosubstrate based on this sequence . other inhibitor molecules can be designed once further phosphorylation sites have been identified . the bifunctional inhibitors could mimic the interactions of both atp and protein / peptide substrates with the ulk3 active site . the inhibitor may also be a peptidomimetic compound or any other molecule that exhibits strong binding affinity to the atp binding site or the protein / peptide substrate - binding site of ulk3 . in the above - described cases , the inhibitor may use similar or different interactions compared to the substrates when bound to ulk3 as far it occupies the substrate binding sites in such a way that the binding of physiological substrates is hindered . alternatively , the inhibitor may be designed to bind the hydrophilic region in the c - terminal non - kinase domain of ulk3 . this region bears sequence and functional homology to the drosophila analog of the kinase and has been shown to be a binding site for the protein interaction partners of the kinase . such inhibitor may also be a multifunctional inhibitor containing both the active site binding moiety and the hydrophilic region binding moiety covalently connected to each other by a linker . for example , this linker may be a synthetic aliphatic linker , or a peptide linker . in case the inhibitor is cell permeable , for example , a small - molecule atp - competitive inhibitor , it can be used as a drug molecule that interferes with the ulk3 regulatory function in the shh pathway . the inhibitor could be designed to contain a specific cell - 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