Dataset Information

AKT and AMPK site-specific phosphorylation on TBC1D4 biological activity

ABSTRACT: TBC1D4 is a 160 kDa multi-domain containing RabGAP (Rab GTPase-activating protein) and a downstream target of the insulin and contraction-activated kinases AKT and AMPK. While phosphorylation of TBC1D4 has been linked to GLUT4 translocation from storage vesicles (GSVs) to the cell surface, its impact on TBC1D4 enzymatic function is not well understood as previous studies mostly investigated the truncated GAP domain lacking the phosphorylation sites. In the present study, we expressed and purified recombinant full-length TBC1D4 using the baculovirus system. Size exclusion chromatography and co-immunoprecipitation experiments revealed that full-length TBC1D4 forms oligomers of ~600 kDa. Moreover, full-length TBC1D4 displayed similar substrate specificity but had a markedly higher specific GAP activity towards Rab10 compared to the truncated GAP domain. Using high-resolution mass spectrometry, we mapped 19 Ser/Thr phosphorylation sites in TBC1D4.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Stefan Lehr

LAB HEAD: Prof. Hadi Al-Hasani

PROVIDER: PXD024645 | Pride | 2021-09-10

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	18102019_8521_TBC1D4_band1.msf	Msf
	18102019_8521_TBC1D4_band1.pep.xml	Pepxml
	18102019_8521_TBC1D4_band1.raw	Raw
	18102019_8522_TBC1D4_band2.msf	Msf
	18102019_8522_TBC1D4_band2.pep.xml	Pepxml

Items per page:

1 - 5 of 37

Publications

AKT/AMPK-mediated phosphorylation of TBC1D4 disrupts the interaction with insulin-regulated aminopeptidase.

Eickelschulte Samaneh S Hartwig Sonja S Leiser Ben B Lehr Stefan S Joschko Viola V Chokkalingam Manopriya M Chadt Alexandra A Al-Hasani Hadi H

The Journal of biological chemistry 20210101

TBC1D4 is a 160 kDa multidomain Rab GTPase-activating protein (RabGAP) and a downstream target of the insulin- and contraction-activated kinases AKT and AMPK. Phosphorylation of TBC1D4 has been linked to translocation of GLUT4 from storage vesicles (GSVs) to the cell surface. However, its impact on enzymatic activity is not well understood, as previous studies mostly investigated the truncated GAP domain lacking the known phosphorylation sites. In the present study, we expressed and purified rec ...[more]

PMID: 33872597

Similar Datasets

Project description:Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 in the N-terminal transactivation domain. In this study, the role of this phosphorylation site was investigated by characterizing the phosphorylation site mutant in the context of full length and truncated AR lacking the ligand-binding domain. The Y267F mutant showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. Expression of AR responsive genes in cells expressing truncated AR wt and AR-Y267F mutant under androgen deprived conditions was assessed by microarray gene expression analysis. The AR pathway score was increased in cells expressing truncated AR wt and decreased in cells expressing truncated AR-Y267F. These results support the concept that phosphorylation of Tyr-267 is required for AR nuclear translocation and recruitment and DNA binding and transcription of AR responsive genes. Gene expression profiling was performed using RNA samples from LNCaP cells expressing truncated AR-wt and truncated AR-Y267F growing in androgen deprived conditions. The RNA sample from vector control cells were used as reference sample. Two biological replicates were used per each cell line.