Src homologous and collagen (Shc) protein binds to F-actin and translocates to the cytoskeleton upon nerve growth factor stimulation in PC12 cells.
Immunoprecipitates of metabolically labeled PC12 cells consistently contained a 43-kDa protein that was associated with Shc, a signal-transducing protein with a single SH2 domain. Following affinity chromatography with immobilized recombinant glutathione S-transferase (GST)-Shc fusion protein, the 43-kDa protein was identified as actin by mass spectrometry and immunoblotting. Cosedimentation experiments using purified actin and GST-Shc showed that Shc binds directly to F-actin, confirming Shc-actin interaction in vivo. Various GST-truncated Shc fusion proteins were prepared and used in actin cosedimentation assays. Constructs containing the SH2 and collagen homology domains were not precipitated, and those containing the amino-terminal domain were. Thus, Shc-actin interactions do not occur in the region of tyrosine phosphorylation and leave the SH2 domain free to bind to other tyrosine-phosphorylated molecules. Although the major pool of Shc in unstimulated PC12 cells is soluble, two other pools are associated with the cytoskeleton and the submembranous cytoskeleton. Upon nerve growth factor stimulation, approximately 50% of the soluble Shc translocates to both cytoskeleton environments within 2 min, decreasing thereafter. When cells were pretreated with cytochalasin D, a drug that disrupts actin filaments, Shc translocation to the cytoskeleton was abolished. However, in the submembranous fraction, the Shc level was elevated in resting cells following cytochalasin D treatment. The kinetics of translocation, compared to mitogen-activated protein kinase activation, and the nature of the Shc-actin interaction suggest that the cytoskeletal association of Shc, induced by growth factors, may be related to membrane ruffling and actin fiber reorganization.
D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D003094:Collagen / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000494:pharmacology*; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D016716:PC12 Cells / Q000187:drug effects* / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D009414:Nerve Growth Factors; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D003094:Collagen; D015688:Oncogene Protein pp60(v-src)