TheDasatinib-Activity

ins .Inaddition,overexpression sociationbetweenactive��1-integrinandpFAKwasobservedonearly
20
2
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a
GFP�C
Rab5-CA
b
GFP�CRab5-CA
FN
Fak�C/�C Ones currentDasatinib-Sport MEFs + GFP�CFAK
?1-integrin
1.2
0.8
0.4
0
Lys
PM
Cyto
Endo
Mr (K)
150
EEA1
Rab21
500 nm
25
150
?1-integrin
GFP
a hundred
one hundred
150
0
0.5
1.0
1.5
?1-integrin
FN
Distance (?m)
GFP�C
Rab5-CA
Talin
GFP�CRab5-CA
pFAK-Y397
FN(7�C10)
Vinculin
Paxillin
Tubulin
a hundred
75
1.2
0.8
0.4
0
500 nm
50
50
ten ?m
0
0.5 1.0 1.5 2.0 2.5
FN(7�C10) pFAK-Y397
Distance (?m)
Figure2 pFAK-Y397 localizes to endosomes together with ?bronectin.


(a) Lively ��1-integrin, pFAK The most importantImatinib-Competition and ?bronectin localization in GFP�CRab5-
CA-expressing TIFFs plated on ?bronectin (best panel) or incubated with
647-labelled ?bronectin fragment (FN(7�C10)) for 45min (bottom panel).
Fluorescenceintensitypro?les(normalizedgreyvalue)acrossrepresentative
endosomes(solidlines)areillustrated.(b)SubcellularfractionationofFak?/?
MEFs re-expressing GFP�CFAK and representative immunoblots from far more
thanthreeindependentexperiments.Lys,celllysate;PM,plasmamembrane;
Cyto,cytosol;Endo,endosomalfraction;ROI,regionofinterest.Unprocessed
originalscansofblotsareshowninSupplementaryFig.9.
endosomeantigen-1(EEA1)-(Fig.1b)andRab21-positiveendosomes Endocytosiscontributestointegrinsignalling
(SupplementaryFig.1e).Forcomparison,themeandistancebetween Following,wesetouttoinvestigatewhethertheendosome-associatedactive
randomlydistributednon-endosomalpFAKandpFAK(Fig.

1cand integrinandFAKarecrucialcomponentsofintegrin�CECM-induced
SupplementaryFig.1e)oractive��1-integrinandpFAKoutsidethe signalling. Serum-starved cells had been Ones pre-existingPomalidomide-Mission maintained in suspension for
endosomes(Fig.1b)was?8�C10pixels.Thesepunctaco-distribution 1h (to inactivate RTKs and adhesion-induced pathways) and
21
indicate that lively ��1-integrin and pFAK are found in subsequently plated on integrin ligands. Below these situations,
thesameendosomes.Phospho-FAKrecruitmenttointegrin-positive ECMengagementinducedtime-dependentactivationofthecanonical
endosomeswasfurtherconfirmedusingsuper-resolutionstimulated downstream integrin-signalling mediators together with FAK, ERK1/2
emissiondepletion(STED)microscopy(Fig.1d).

Thus,phosphory- andAktwithoutsignificantadhesion-dependentactivationofanyof
latedFAKisnotrestrictedtofocaladhesionsbutcanalsobedetected the 48 di?erent RTKs tested (Supplementary Fig. 2a,b), suggesting
analyses
onendosomesinadherentcells.
that the observed signals had been a consequence of integrin-mediated
adhesionandnotduetogrowthfactorreceptorstimulation.Dynasore-
Integrinligandsandtalinarepresentin
FAK-positiveendosomes
mediatedinhibitionofdynamin
receptor endocytosis , abrogated integrin and transferrin receptor
internalization(SupplementaryFig.3a�Cc)andsignificantlyattenuated
24
,animportantmediatorofintegrin
sixteen
Severalstudiesreportintegrinligandlocalizationinendosomes
8,ten,22
and we've got demonstrated greater endosomal retention of theactivationofFAKandERK1/2incarcinomacellsandinnormal
active ��1