The N terminus controls sterol binding while the C terminus regulates the scaffolding function of OSBP.
Previously we reported that when cell cholesterol is acutely lowered with beta-methyl-cyclodextrin the amount of activated ERK1/2 in caveolae dramatically increases. We traced the origin of this novel method of pERK1/2 accumulation to a macromolecular complex with dual specific phosphatase activity that contains the serine/threonine phosphatase PP2A, the tyrosine phosphatase HePTP, the oxysterol-binding protein OSBP and cholesterol. When cell cholesterol is lowered, or oxysterols is introduced, the complex disassembles and pERK1/2 increases. In an effort to better understand how OSBP functions as a cholesterol-regulated scaffolding protein, we have mapped the functional parts of the molecule. The command center of the molecule is a centrally located, 51 amino acids (408-459) long sterol-binding domain that can bind both cholesterol and 25-hydroxycholesterol. This domain is functional whether attached to the N- or the C-terminal half of OSBP. Introduction of a Y458S mutation impairs binding. Even though 25-hydroxycholesterol will compete for cholesterol binding to OSBP(408-809), it will not compete for cholesterol binding in full-length OSBP. Upon further analysis we found that a glycine-alaninerich region at the N-terminal end of OSBP works with the PH domain to control cholesterol binding without affecting 25-hydroxycholesterol binding. Finally, we found that HePTP and PP2A bind the C-terminal half of OSBP, HePTP binds a coiled-coil domain (amino acids 732-761), and PP2A binds neither the coiled-coil nor HePTP. On the basis of this information we propose a new model for how OSBP is able to sense both membrane cholesterol and oxidized sterols and link this information to the ERK1/2 signaling pathway.
Wang
Ping-Yuan
PY
Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9039, USA.
Weng
Jian
J
Lee
Sungsoo
S
Anderson
Richard G W
RG
D019943:Amino Acid Substitution; D000818:Animals; D006367:HeLa Cells; D006801:Humans; D006888:Hydroxycholesterols / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010449:Peptide Mapping; D011485:Protein Binding / Q000235:genetics; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D054558:Protein Tyrosine Phosphatases, Non-Receptor / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D000098825:Oxysterol Binding Proteins
D006888:Hydroxycholesterols; D011987:Receptors, Steroid; D000098825:Oxysterol Binding Proteins; C007997:25-hydroxycholesterol; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D054648:Protein Phosphatase 2; C502159:PTPN7 protein, human; D054558:Protein Tyrosine Phosphatases, Non-Receptor