The 45-kDa form of Pdx-1 does not result from post-translational modifications.
Pdx-1 is a key regulator of glucose-stimulated insulin gene transcription in beta-cells. The regulation of Pdx-1 in response to glucose has previously been associated with a remarkable shift in electrophoretic mobility on SDS-PAGE from 31 to 45kDa. This has been attributed to different post-translational modifications including phosphorylation, sumoylation or glycosylation. However, and in contrast with previous studies, we describe in this paper that Pdx-1 produced in Escherichia coli, by in vitro transcription/translation or exogenously expressed in eukaryotic cells, migrates with an apparent molecular mass of 45kDa despite a calculated mass of 31kDa. Moreover, we show that the migration of endogenous Pdx-1 obtained from a mouse beta-cell line as well as from human primary islets is not dependent on glucose concentration. Taken together, these data, validated by mass spectrometry techniques, establish that anomalous migration of Pdx-1 on SDS-PAGE does not result from post-translational modifications.
Carlotti
Françoise
F
Department of Molecular Cell Biology-Virus and Stem Cell Biology Laboratory, Leiden University Medical Center, Postal Zone S1-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands. F.Carlotti@lumc.n
Zaldumbide
Arnaud
A
Charif
Halima
H
de Koning
Eelco J
EJ
Luider
Theo M
TM
Hoeben
Rob C
RC
D000818:Animals; D016477:Artifacts; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D018398:Homeodomain Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D005947:Glucose