SUPPLEMENTARY INFORMATION

Transcription

1 DOI: 1.138/nc2274 EpH4 Prentl -/MDCK EpH4 Prentl -/MDCK - FERM- Figure S1 (kd) delferm- (1-438)- c Prentl MDCK -/MDCK deljfr- Input Control IP IP Input Control IP IP d Control Control Figure S1 () Specificity of ntiody nd endogenous expression of. Western lotting for or ws performed ginst cell homogentes from EpH4, MDCK nd -/MDCK cells. ntiody specificlly recognized endogenous protein in EpH4 cells nd exogenous - protein in -/MDCK cells. Endogenous expression ws hrdly detected in MDCK cells. () JFR of is necessry to recruit to the AJC. Deletion mutnts of were expressed in MDCK cells, nd these cells were immunostined for nd. Scle r, 5 μm. (c) does not ffect - interctions. ws immunoprecipitted from prentl or -/MDCK cells, nd the precipittes were immunolotted for or. (d) Depletion of or does not ffect the locliztion nd expression of the other. EpH4 cells were trnsfected with control, or RNA expression vector, together with. Cells were doule-immunostined for nd or. Scle r, 5 μm. 1

2 Figure S2 si si si si si PKCλ signl intensity (A.U.) * si si si DKD si PKCλ H2O Y si si H2O Y signl intensity (A.U.) H2O Y si H2O Y si si si 2P-MLC 2P-MLC 2P-MLC 2P-MLC 2P-MLC signl intensity (A.U.) H2O Y si H2O Y si Figure S2 () Upregultion of myosin IIB t AJCs in, or PKCλ knockdown cells. These molecules were depleted y sirna in EpH4 cells. Cells were immunostined for nd myosin IIB (). Scle r, 5 μm. Signl intensity of myosin IIB t AJCs in knocked down EpH4 cells is indicted in the grph, which ows representtive result of three independent experiments. Asterisks on rs indicte tht the men is significntly different from si control (men ± s.e.m, *p<.5, p<.1, Student s t-test, n>2 cells). () Inhiition of ROCK ctivity ttenutes DKDinduced upregultion of ctomyosin ctivity. nd were depleted y sirna in EpH4 cells. After the tretment with or without Y-27632, cells were immunostined for nd myosin IIB or 2P-MLC (pt18/ps19- MLC). Scle r, 5 μm. Signl intensity of myosin IIB or 2P-MLC t AJCs in knocked down EpH4 cells with or without Y tretment is indicted in the grph (men ± s.e.m, p<.1, Student s t-test, n>2 cells). 2

3 Prentl MDCK c Figure S3 si Cont-1 si Cont-2 si -1 si -2 si -/MDCK si PKCλ Signl intensity (A.U.) Prentl - d si si 2 Apicl re (µm 2 ) si si si si si Figure S3 ehves like. () Prentl MDCK nd -/ MDCK cells immunostined for nd. Scle r, 5 μm. Signl intensity of t AJCs is indicted in the grph (men ±s.e.m, p<.1, Student s t-test, n>2 cells). (), or oth of them were depleted y sirna in EpH4 cells. Cells were immunostined for nd. Locliztion of t AJCs indicted y rrows is mgnified t the right pnels. Scle r, 5 μm. (c) or ws depleted in EpH4 cells, nd these proteins were detected y doule immunostining. Ded lines indicte the orders etween norml nd KD cells. Scle r, 5 μm. Knockdown efficiency for is lso indicted y Western lotting. Knockdown of does not ffect, or protein level. (d) ws depleted in EpH4 cells. Cells were immunostined for, or myosin IIB. Ded lines indicte the orders etween norml nd KD cells. Scle r, 5 μm. Apicl re of ech cell ws mesured using -stined smples (men ± s.e.m, p<.1, Student s t-test, n>3 cells). 3

4 Control 1 Control 2 #27 # Control 1 MDCK #27 MDCK Control 1 MDCK #27 MDCK Apicl re (µm 2 ) 2,5 2, 1,5 1, 5 Control 1 #27 # P-MLC signl intensity (A.U.) Control 1 #27 c P-MLC signl intensity (A.U.) ** delbd - - deljfr - - Figure S4 Stle KD MDCK cells ow picl constriction nd upregultion of P-MLC. () MDCK cells stly expressing control or RNA were generted, nd their expression levels of were exmined y Western lotting. #27 or # indictes individul clone. Cells were immunostined for. Scle r, 5 μm. () Cells were immunostined for, or. The regions surrounded y ded squre re mgnified t right pnels. signls visile in the control cells re minly derived from stress fiers ut not from AJCs. Scle r, 5 μm. Apicl re of ech cell or signl intensity t AJCs is indicted in the grphs, ech of which ows representtive result of three independent experiments. Asterisks on rs indicte tht the men is significntly different from control 1 (men ± s.e.m, p<.1, Student s t-test, n>5 cells). (c) Signl intensity of 1P- MLC t AJCs in #27 MDCK cells expressing the indicted proteins. Grph ows representtive result of three independent experiments. Asterisks indicte tht the men is significntly different from control (men ± s.e.m, **p<.1, p<.1, Student s t-test, n>2 cells). 4

5 GAP- /EpH4 si si GAP-PKCι- /EpH4 si si JAMA- /EpH4 si si JAMA-PKCι- /EpH4 si si Figure S5 Junctionl locliztion of is sufficient to inhiit the upregultion of P-MLC induced y DKD of nd. nd were depleted in stle EpH4 trnsfectnts expressing GAP-, GAP-PKCι-, JAMA- or JAMA-PKCι-. Cells were immunostined for, nd. Scle r, 5 μm. 5

6 endogenous S417,T124,S1163,T1189,S1281,S13,T14, T17,S1341 MSTGDSFETRFEKIDNLLRDPKSEVNSDCLLDGLDALVYDLDFPALRKNK NIDNFLSRYKDTINKIRDLRMKAEDYEVVKVIGRGAFGEVQLVRHKSTRK VYAMKLLSKFEMIKRSDSAFFWEERDIMAFANSPWVVQLFYAFQDDRYLY MVMEYMPGGDLVNLMSNYDVPEKWARFYTAEVVLALDAIHSMGFIHRDVK PDNMLLDKSGHLKLADFGTCMKMNKEGMVRCDTAVGTPDYISPEVLKSQG GDGYYGRECDWWSVGVFLYEMLVGDTPFYADSLVGTYSKIMNHKNSLTFP DDNDISKEAKNLICAFLTDREVRLGRNGVEEIKRHLFFKNDQWAWETLRD TVAPVVPDLSSDIDTSNFDDLEEDKGDEETFPIPKAFVGNQLPFVGFTYY SNRRYLPSANASENRSSSNVDKSLQESLQKTIYKLEEQLHNEMQLKDEME QKCRTSNLKLDKIMKELDEEGNQRRNLESAVSQIEKEKMLLQHRINEYQR KVEQENEKRRNIENEVSTLKDQLEDLRKASQTSQLANEKLTQLQKQLEEA NDLLRTESDTAVRLRKSHTEMSKSISQLESLNRELQERNRILENSKSQAD KDYYQLQAVLEAERRDRGHDSEMIGDLQARITSLQEEVKHLKHNLERVEG ERKEAQDMLNHSEKEKNNLEIDLNYKLKSIQQRLEQEVNEHKVTKARLTD KHQSIEEAKSVAMCEMEKKLKEEREAREKAENRVVETEKQCSMLDVDLKQ SQQKLEHLTENKERMEDEVKNLALQLEQESNKRLLLQNELKTQAFEADNL KGLEKQMKQEINTLLEAKRLLEFELAQLTKQYRGNEGQMRELQDQLEAEQ YFSTLYKTQVKELKEEIEEKNRENLRKIQELQSEKETLSTQLDLAETKAE SEQLARGILEEQYFELTQESKKAASRNRQEITDKDHTVSRLEETNSVLTK DIEMLRKENEELNERMRTAEEEYKLKKEEEINNLKAAFEKNISTERTLKT QAVNKLAEIMNRKDFKIDRKKANTQDLRKKEKENRKLQLELNQEREKFNQ MVVKHQKELNDMQAQLVEECTHRNELQMQLASKESDIEQLRAKLLDLSDS TSVASFPSADETDGNLPESRIEGWLSVPNRGNIKRYGWKKQYVVVSSKKI LFYNDEQDKEQSSPSMVLDIDKLFHVRPVTQGDVYRAETEEIPKIFQILY ANEGECRKDIEVEPVQQGEKTNFQNHKGHEFIPTLYHFPANCEACAKPLW HVFKPPPALECRRCHVKCHRDHLDKKEDLISPCKVSYDVTSARDMLLLAC SQDEQKKWVTHLVKKIPKNPPSGFVRASPRTLSTRSTANQSFRKVVKNTS GKTS in vitro kinse ssy T219,S679,T997,T1,T124,S1291,S131,S1322, S13,T14,T17,S1341 MSTGDSFETRFEKIDNLLRDPKSEVNSDCLLDGLDALVYDLDFPALRKNK NIDNFLSRYKDTINKIRDLRMKAEDYEVVKVIGRGAFGEVQLVRHKSTRK VYAMKLLSKFEMIKRSDSAFFWEERDIMAFANSPWVVQLFYAFQDDRYLY MVMEYMPGGDLVNLMSNYDVPEKWARFYTAEVVLALDAIHSMGFIHRDVK PDNMLLDKSGHLKLADFGTCMKMNKEGMVRCDTAVGTPDYISPEVLKSQG GDGYYGRECDWWSVGVFLYEMLVGDTPFYADSLVGTYSKIMNHKNSLTFP DDNDISKEAKNLICAFLTDREVRLGRNGVEEIKRHLFFKNDQWAWETLRD TVAPVVPDLSSDIDTSNFDDLEEDKGDEETFPIPKAFVGNQLPFVGFTYY SNRRYLPSANASENRSSSNVDKSLQESLQKTIYKLEEQLHNEMQLKDEME QKCRTSNLKLDKIMKELDEEGNQRRNLESAVSQIEKEKMLLQHRINEYQR KVEQENEKRRNIENEVSTLKDQLEDLRKASQTSQLANEKLTQLQKQLEEA NDLLRTESDTAVRLRKSHTEMSKSISQLESLNRELQERNRILENSKSQAD KDYYQLQAVLEAERRDRGHDSEMIGDLQARITSLQEEVKHLKHNLERVEG ERKEAQDMLNHSEKEKNNLEIDLNYKLKSIQQRLEQEVNEHKVTKARLTD KHQSIEEAKSVAMCEMEKKLKEEREAREKAENRVVETEKQCSMLDVDLKQ SQQKLEHLTENKERMEDEVKNLALQLEQESNKRLLLQNELKTQAFEADNL KGLEKQMKQEINTLLEAKRLLEFELAQLTKQYRGNEGQMRELQDQLEAEQ YFSTLYKTQVKELKEEIEEKNRENLRKIQELQSEKETLSTQLDLAETKAE SEQLARGILEEQYFELTQESKKAASRNRQEITDKDHTVSRLEETNSVLTK DIEMLRKENEELNERMRTAEEEYKLKKEEEINNLKAAFEKNISTERTLKT QAVNKLAEIMNRKDFKIDRKKANTQDLRKKEKENRKLQLELNQEREKFNQ MVVKHQKELNDMQAQLVEECTHRNELQMQLASKESDIEQLRAKLLDLSDS TSVASFPSADETDGNLPESRIEGWLSVPNRGNIKRYGWKKQYVVVSSKKI LFYNDEQDKEQSSPSMVLDIDKLFHVRPVTQGDVYRAETEEIPKIFQILY ANEGECRKDIEVEPVQQGEKTNFQNHKGHEFIPTLYHFPANCEACAKPLW HVFKPPPALECRRCHVKCHRDHLDKKEDLISPCKVSYDVTSARDMLLLAC SQDEQKKWVTHLVKKIPKNPPSGFVRASPRTLSTRSTANQSFRKVVKNTS GKTS coverge(~6%) Figure S6 Determintion of phosphoryltion sites y LC-MS/MS. All phosphoryltion sites identified y endogenous immunoprecipittion nd in vitro kinse ssy re indicted on top. The full-length mino cid sequence of is own, nd the sequences covered y LC-MS/MS re indicted in red letters. Phosphoryltion sites commonly identified in oth ssys re underlined in lue. 6

7 MDCK cells Control 1 Control 2 ROCKs #11 ROCKs #15, ROCK2 ROCKs, c Y (-) Y () si control #1 si control #2 si PKCλ #1 si PKCλ #2 si control #1 si control #2 si PKCλ #1 si PKCλ #2 GST-MLC (~47 kd) GST (GST-MLC) CBB (IP) d incution time 1' 5' 15' Y GST rec.pkcι e GST (GST-MLC) (IP) WT 4A 4E 5A 5E GST (GST-MLC) - (IP) (IP) f g Pull down GST GTPγS-GST-RhoA Input µg GST-RhoA (~ kd) - WT CBB IB: Input WT 4A 4E 5A 5E GTPγS-GST-RhoA-pull down (2µg) WT 4A 4E 5A 5E - IB: Figure S7 () or ROCKs knockdown in MDCK cells. RNA expression vector for or ROCKs were co-trnsfected with expression vector. Cells were immunostined for nd. Scle r, 5 μm. MDCK cells stly expressing control or ROCKs RNA were generted, nd their expressions of nd ROCK2 were exmined y Western lotting. #11 or #15 indictes individul clone. (-g) Phosphoryltion of y does not ffect the kinse ctivity of or Rho- interctions. () GST- MLC ws prepred from E. coli, nd stined with CBB. (c) Immunoprecipitted fter PKCλ knockdown ws used to perform immune complex kinse ssy ginst GST-MLC with or without Y Kinse ctivity ws estimted y Western lotting for. (d) Immunoprecipitted ws incuted with GST (control) or recominnt (rec.) PKCι for phosphoryltion. After GST or rec. PKCι ws wed out, immunoprecipitted ws used to perform immune complex kinse ssy ginst GST-MLC with or without Y for indicted times. Kinse ctivity ws estimted y Western lotting for. (e), wild type, unphosphoryltle or phosphomimetic mutnts of - ws expressed in HEK293 cells nd immunoprecipitted with ntiody. Immunoprecipitted proteins were used to perform immune complex kinse ssy ginst GST-MLC. Kinse ctivity ws estimted y Western lotting for. (f) GST-RhoA ws prepred from E. coli, nd stined with CBB. (g) Wild type - ws expressed in HEK293 cells, nd pull-down ssy ws performed with different mounts of GTPγS-GST-RhoA or GST to determine the unsturted concentrtion of GTPγS-GST-RhoA, which cn ind -., wild type, unphosphoryltle or phosphomimetic mutnts of - were expressed in HEK293 cells, nd pulled down with 2 μg of GTPγS-GST-RhoA. 7

8 -C3 ( #27 MDCK) Figure S8 Rho ctivity is required for the mintennce of AJCs. or -C3 (C3, otulinum C3 toxin; n inhiitor of Rho ctivity) ws expressed in #27 MDCK cells. Cells were immunostined for nd. signls re missing in the cells expressing -C3. Scle r, 5 μm. 8

9 Fig. 1 Fig. 1 Fig. 2 Fig. 1d Fig. 3 Fig. 3d Fig. 4 Fig. 4 Fig. 4e Fig. 5c Fig. S1 Fig. S3 Fig. S7c Fig. S12 Flg Fig. S12c GST Fig. S8 Fig. S12d GAPDH N-cdherin GST Fig. S11 Fig. S12f - () ROCK2 GST Figure S9 Full scns of Western lots presented. The red oxes indicte the res used in the figures. 9