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1.
Mol Cell Biol ; 35(9): 1506-22, 2015 May.
Article in English | MEDLINE | ID: mdl-25733683

ABSTRACT

Cyclin D and cyclin-dependent kinase 4 (cdk4) are overexpressed in a variety of tumors, but their levels are not accurate indicators of oncogenic activity because an accessory factor such as p27(Kip1) is required to assemble this unstable dimer. Additionally, tyrosine (Y) phosphorylation of p27 (pY88) is required to activate cdk4, acting as an "on/off switch." We identified two SH3 recruitment domains within p27 that modulate pY88, thereby modulating cdk4 activity. Via an SH3-PXXP interaction screen, we identified Brk (breast tumor-related kinase) as a high-affinity p27 kinase. Modulation of Brk in breast cancer cells modulates pY88 and increases resistance to the cdk4 inhibitor PD 0332991. An alternatively spliced form of Brk (Alt Brk) which contains its SH3 domain blocks pY88 and acts as an endogenous cdk4 inhibitor, identifying a potentially targetable regulatory region within p27. Brk is overexpressed in 60% of breast carcinomas, suggesting that this facilitates cell cycle progression by modulating cdk4 through p27 Y phosphorylation. p27 has been considered a tumor suppressor, but our data strengthen the idea that it should also be considered an oncoprotein, responsible for cyclin D-cdk4 activity.


Subject(s)
Breast Neoplasms/enzymology , Cyclin D/metabolism , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Neoplasm Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Breast/enzymology , Cell Line, Tumor , Female , Humans , Neoplasm Proteins/chemistry , Phosphorylation , Protein Interaction Maps , Protein-Tyrosine Kinases/chemistry , src Homology Domains
2.
J Cell Biol ; 194(6): 873-87, 2011 Sep 19.
Article in English | MEDLINE | ID: mdl-21911479

ABSTRACT

The epithelial cell-specific clathrin adaptor complex AP-1B facilitates the sorting of various transmembrane proteins from recycling endosomes (REs) to the basolateral plasma membrane. Despite AP-1B's clear importance in polarized epithelial cells, we still do not fully understand how AP-1B orchestrates basolateral targeting. Here we identify the ADP-ribosylation factor 6 (Arf6) as an important regulator of AP-1B. We show that activated Arf6 pulled down AP-1B in vitro. Furthermore, interfering with Arf6 function through overexpression of dominant-active Arf6Q67L or dominant-negative Arf6D125N, as well as depletion of Arf6 with short hairpin RNA (shRNA), led to apical missorting of AP-1B-dependent cargos. In agreement with these data, we found that Arf6 colocalized with AP-1B and transferrin receptor (TfnR) in REs. In addition, we observed specific recruitment of AP-1B into Arf6-induced membrane ruffles in nonpolarized cells. We conclude that activated Arf6 directs membrane recruitment of AP-1B, thus regulating AP-1B's functions in polarized epithelial cells.


Subject(s)
ADP-Ribosylation Factors/genetics , Adaptor Protein Complex 1/metabolism , Cell Polarity/physiology , Epithelial Cells/metabolism , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/metabolism , Adaptor Protein Complex 1/genetics , Animals , Cells, Cultured , Dogs , Epithelial Cells/cytology , Fluorescent Antibody Technique , HEK293 Cells , Humans , Mice , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Receptors, Transferrin/genetics , Receptors, Transferrin/metabolism , Swine
3.
Mol Biol Cell ; 21(1): 95-105, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19864464

ABSTRACT

Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell-specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits micro1A or micro1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of micro1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in micro1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P(3) formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B-dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P(3) formation in recycling endosomes is essential for AP-1B function.


Subject(s)
Adaptor Protein Complex mu Subunits/metabolism , Cell Polarity , Endocytosis , Endosomes/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Phosphatidylinositol Phosphates/metabolism , Adaptor Protein Complex mu Subunits/chemistry , Amino Acid Sequence , Amino Acids/metabolism , Animals , Cell Line , Gene Knockdown Techniques , Green Fluorescent Proteins/metabolism , Humans , Mice , Molecular Sequence Data , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Transport , Receptors, Transferrin/metabolism , Recombinant Fusion Proteins/metabolism , Swine
4.
J Cell Biol ; 177(3): 477-88, 2007 May 07.
Article in English | MEDLINE | ID: mdl-17485489

ABSTRACT

The epithelial cell-specific adaptor complex AP-1B is crucial for correct delivery of many transmembrane proteins from recycling endosomes to the basolateral plasma membrane. Subsequently, membrane fusion is dependent on the formation of complexes between SNARE proteins located at the target membrane and on transport vesicles. Although the t-SNARE syntaxin 4 has been localized to the basolateral membrane, the v-SNARE operative in the AP-1B pathway remained unknown. We show that the ubiquitously expressed v-SNARE cellubrevin localizes to the basolateral membrane and to recycling endosomes, where it colocalizes with AP-1B. Furthermore, we demonstrate that cellubrevin coimmunoprecipitates preferentially with syntaxin 4, implicating this v-SNARE in basolateral fusion events. Cleavage of cellubrevin with tetanus neurotoxin (TeNT) results in scattering of AP-1B localization and missorting of AP-1B-dependent cargos, such as transferrin receptor and a truncated low-density lipoprotein receptor, LDLR-CT27. These data suggest that cellubrevin and AP-1B cooperate in basolateral membrane trafficking.


Subject(s)
Adaptor Protein Complex 1/metabolism , Adaptor Protein Complex beta Subunits/metabolism , Cell Polarity/physiology , Endosomes/metabolism , Epithelial Cells/metabolism , SNARE Proteins/metabolism , Vesicle-Associated Membrane Protein 3/metabolism , Adaptor Protein Complex 1/genetics , Adaptor Protein Complex beta Subunits/genetics , Animals , Cell Line , Cell Membrane/metabolism , Cell Polarity/drug effects , Dogs , Epithelial Cells/cytology , Humans , Membrane Fusion/drug effects , Membrane Fusion/physiology , Metalloendopeptidases/pharmacology , Protein Transport/drug effects , Protein Transport/physiology , Qa-SNARE Proteins/genetics , Qa-SNARE Proteins/metabolism , Receptors, LDL/metabolism , SNARE Proteins/genetics , Tetanus Toxin/pharmacology , Vesicle-Associated Membrane Protein 3/genetics
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