title: Proteomic profiling of endothelial invasion revealed receptor for activated C kinase 1 (RACK1) complexed with vimentin to regulate focal adhesion kinase (FAK).
abstract: Angiogenesis is critical for many physiological and pathological processes. To identify molecules relevant to angiogenesis, we performed a proteomic screen comparing invading versus non-invading endothelial cells in three-dimensional collagen matrices. We found up-regulated levels of receptor for activated C kinase 1 (RACK1) and the intermediate filament protein vimentin that correlated with increased endothelial cell invasion. Because both RACK1 and vimentin have been linked to focal adhesion kinase (FAK), we investigated whether this pathway regulated invasion. RACK1 depletion reduced invasion responses, and this was associated with attenuated activation of FAK. Knockdown of vimentin significantly decreased levels of phosphorylated and total FAK. Treatment with a pharmacological inhibitor of FAK dose-dependently reduced invasion, indicating a crucial role for FAK activity during invasion. Because RACK1 and vimentin were both up-regulated with sphingosine 1-phosphate treatment, required for invasion, and regulated FAK, we tested whether they complexed together. RACK1 complexed with vimentin, and growth factors enhanced this interaction. In addition, RACK1, vimentin, and FAK formed an intermolecular complex in invading endothelial cultures in three dimensions in response to stimulation by sphingosine 1-phosphate and growth factors. Moreover, depletion of RACK1 decreased the association of vimentin and FAK, suggesting that RACK1 was required for stabilizing vimentin-FAK interactions during sprouting. Silencing of vimentin and RACK1 decreased cell adhesion and focal contact formation. Taken together, these results demonstrate that proangiogenic signals converge to enhance expression and association of RACK1 and vimentin, which regulated FAK, resulting in successful endothelial sprout formation in three-dimensional collagen matrices.
authors.affiliation: From the Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas 77843.
authors.forename: Jui M
authors.initials: JM
authors.lastname: Dave
authors.affiliation: From the Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas 77843.
authors.forename: Hojin
authors.initials: H
authors.lastname: Kang
authors.affiliation: From the Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas 77843.
authors.forename: Colette A
authors.initials: CA
authors.lastname: Abbey
authors.affiliation: From the Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas 77843.
authors.forename: Steve A
authors.initials: SA
authors.lastname: Maxwell
authors.affiliation: From the Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas 77843. Electronic address: kbayless@medicine.tamhsc.edu.
authors.forename: Kayla J
authors.initials: KJ
authors.lastname: Bayless
mesh_terms: D003094:Collagen / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D055785:Gene Knockdown Techniques; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008246:Lysophospholipids / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000502:physiology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D055550:Protein Stability; D040901:Proteomics; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology; D014746:Vimentin / Q000235:genetics / Q000378:metabolism*; D000099157:Sphingosine Kinase
chemical_list: D003094:Collagen; D051417:Focal Adhesion Kinase 1; D019204:GTP-Binding Proteins; D008246:Lysophospholipids; D046912:Multiprotein Complexes; D009363:Neoplasm Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D013110:Sphingosine; D014746:Vimentin; D000099157:Sphingosine Kinase; C419368:RACK1 protein, human; C060506:sphingosine 1-phosphate; C496050:PTK2 protein, human
keywords: Angiogenesis; Collagen; Endothelial Cell; Focal Adhesion Kinase; Morphogenesis; RACK1; Vimentin