title: Conformation-Dependent Human p52Shc Phosphorylation by Human c-Src.
abstract: Phosphorylation of the human p52Shc adaptor protein is a key determinant in modulating signaling complex assembly in response to tyrosine kinase signaling cascade activation. The underlying mechanisms that govern p52Shc phosphorylation status are unknown. In this study, p52Shc phosphorylation by human c-Src was investigated using purified proteins to define mechanisms that affect the p52Shc phosphorylation state. We conducted biophysical characterizations of both human p52Shc and human c-Src in solution as well as membrane-mimetic environments using the acidic lipid phosphatidylinositol 4-phosphate or a novel amphipathic detergent (2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside). We then identified p52Shc phosphorylation sites under various solution conditions, and the amount of phosphorylation at each identified site was quantified using mass spectrometry. These data demonstrate that the p52Shc phosphorylation level is altered by the solution environment without affecting the fraction of active c-Src. Mass spectrometry analysis of phosphorylated p52Shc implies functional linkage among phosphorylation sites. This linkage may drive preferential coupling to protein binding partners during signaling complex formation, such as during initial binding interactions with the Grb2 adaptor protein leading to activation of the Ras/MAPK signaling cascade. Remarkably, tyrosine residues involved in Grb2 binding were heavily phosphorylated in a membrane-mimetic environment. The increased phosphorylation level in Grb2 binding residues was also correlated with a decrease in the thermal stability of purified human p52Shc. A schematic for the phosphorylation-dependent interaction between p52Shc and Grb2 is proposed. The results of this study suggest another possible therapeutic strategy for altering protein phosphorylation to regulate signaling cascade activation.
authors.affiliation: †Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Tsutsui
authors.affiliation: †Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Johnson
authors.affiliation: ‡Department of Biochemistry, The University of Texas Health Sciences Center at San Antonio, 7750 Floyd Curl Drive, San Antonio, Texas 78229-3900, United States.
authors.forename: Borries
authors.initials: B
authors.lastname: Demeler
authors.affiliation: ∥Department of Geriatric Medicine, Reynolds Oklahoma Center on Aging, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Kinter
authors.affiliation: †Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.|⊥Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.|∇Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Franklin A
authors.initials: FA
authors.lastname: Hays
mesh_terms: D000081247:CSK Tyrosine-Protein Kinase; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051380:GRB2 Adaptor Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry; D010766:Phosphorylation / Q000502:physiology; D055550:Protein Stability; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019061:src-Family Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D018129:Phosphatidylinositol Phosphates; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; C037178:phosphatidylinositol 4-phosphate; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D048049:Extracellular Signal-Regulated MAP Kinases; D016283:Proto-Oncogene Proteins p21(ras)