title: Functional proteomic analysis of long-term growth factor stimulation and receptor tyrosine kinase coactivation in Swiss 3T3 fibroblasts.
abstract: In Swiss 3T3 fibroblasts, long-term stimulation with PDGF, but not insulin-like growth factor 1 (IGF-1) or EGF, results in the establishment of an elongated migratory phenotype, characterized by the formation of retractile dendritic protrusions and absence of actin stress fibers and focal adhesion complexes. To identify receptor tyrosine kinase-specific reorganization of the Swiss 3T3 proteome during phenotypic differentiation, we compared changes in the pattern of protein synthesis and phosphorylation during long-term exposure to PDGF, IGF-1, EGF, and their combinations using 2DE-based proteomics after (35)S- and (33)P-metabolic labeling. One hundred and five differentially regulated proteins were identified by mass spectrometry and some of these extensively validated. PDGF stimulation produced the highest overall rate of protein synthesis at any given time and induced the most sustained phospho-signaling. Simultaneous activation with two or three of the growth factors revealed both synergistic and antagonistic effects on protein synthesis and expression levels with PDGF showing dominance over both IGF-1 and EGF in generating distinct proteome compositions. Using signaling pathway inhibitors, PI3K was identified as an early site for signal diversification, with sustained activity of the PI3K/AKT pathway critical for regulating late protein synthesis and phosphorylation of target proteins and required for maintaining the PDGF-dependent motile phenotype. Several proteins were identified with novel PI3K/Akt-dependent synthesis and phosphorylations including eEF2, PRS7, RACK-1, acidic calponin, NAP1L1, Hsp73, and fascin. The data also reveal induction/suppression of key F-actin and actomyosin regulators and chaperonins that enable PDGFR to direct the assembly of a motile cytoskeleton, despite simultaneous antagonistic signaling activities. Together, the study demonstrates that long-term exposure to different growth factors results in receptor tyrosine kinase-specific regulation of relatively small subproteomes, and implies that the strength and longevity of receptor tyrosine kinase-specific signals are critical in defining the composition and functional activity of the resulting proteome.
authors.affiliation: Discovery Research Department, Chugai Pharmaceutical Co. Ltd., Japan.
authors.forename: Kohji
authors.initials: K
authors.lastname: Nagano
authors.forename: Akunna
authors.initials: A
authors.lastname: Akpan
authors.forename: Gayathri
authors.initials: G
authors.lastname: Warnasuriya
authors.forename: Steven
authors.initials: S
authors.lastname: Corless
authors.forename: Nick
authors.initials: N
authors.lastname: Totty
authors.forename: Alice
authors.initials: A
authors.lastname: Yang
authors.forename: Robert
authors.initials: R
authors.lastname: Stein
authors.forename: Marketa
authors.initials: M
authors.lastname: Zvelebil
authors.forename: Allan
authors.initials: A
authors.lastname: Stensballe
authors.forename: Al
authors.initials: A
authors.lastname: Burlingame
authors.forename: Michael
authors.initials: M
authors.lastname: Waterfield
authors.forename: Rainer
authors.initials: R
authors.lastname: Cramer
authors.forename: John F
authors.initials: JF
authors.lastname: Timms
authors.forename: Søren
authors.initials: S
authors.lastname: Naaby-Hansen
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005347:Fibroblasts; D005419:Flavonoids / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007553:Isotope Labeling; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D056490:Nucleosome Assembly Protein 1 / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D020543:Proteome / Q000032:analysis*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D001549:Benzamides; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002867:Chromones; D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor; D005419:Flavonoids; D007334:Insulin-Like Growth Factor I; D009025:Morpholines; D056490:Nucleosome Assembly Protein 1; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010982:Platelet-Derived Growth Factor; D020543:Proteome; D051057:Proto-Oncogene Proteins c-akt; D020794:Receptor Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; C120227:2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; C533558:Nap1l1 protein, mouse; C519530:insulin-like growth factor-1, mouse; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one