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.
Highlights
Receptor tyrosine kinases transduce key extracellular signals and trigger multiple cellular events, including proliferation, differentiation, and cytoskeletal rearrangement
The disruption of stress fibers and focal adhesions occurred within the first hour of platelet-derived growth factor (PDGF) stimulation, while full development of dendritic protrusions appeared after 12 hours and was sustained throughout the rest of the stimulation period (Figure 1B)
The PDGF-specific morphological transformation was abrogated in fibroblasts where DNA-dependent RNA-synthesis was blocked by treatment with actinomycin D, protein translation was blocked by cycloheximide treatment, the 26S proteasome was inhibited by PS341 treatment, where the activity of Src kinases was inhibited by SU6656 treatment, and where methyltransferase and deacetylase activities were blocked by 5aC and trichostatin A (TSA) treatments, respectively (Supplementary Data; Figure S2). These findings demonstrate that the PDGFinduced reorganization of the actin cytoskeleton in Swiss 3T3 fibroblasts involves the complex regulation of gene transcription, protein synthesis, post-translational modification and degradation
Summary
Receptor tyrosine kinases transduce key extracellular signals and trigger multiple cellular events, including proliferation, differentiation, and cytoskeletal rearrangement. A variety of intracellular signaling molecules associate with the phosphorylated tyrosine residues on the cytoplasmic tail of activated RTKs via their SH2 or PTB domains Mutation of these docking sites results in severely disturbed cellular phenotypes in different model systems, demonstrating the physiological importance of RTK signaling [1]. Distinct responses to RTKs in different cells can be explained by differences in the cells’ developmental histories These results suggest that RTKs utilize general signaling pathways or networks with some redundancy, and that the specificity provided by combinatorial recruitment of particular signaling proteins is not that stringent. These studies only deal with transcriptional responses to RTK activation, and gene expression changes alone are unlikely to determine biological outcome. Global analyses of protein synthesis, expression level, activity status and degradation rates are necessary to understand how signals from different RTKs are diversified to provide specific biological outcomes
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