Lombardo C R, Consler T G, Kassel D B
Department of Protein Biochemistry, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina 27709, USA.
Biochemistry. 1995 Dec 19;34(50):16456-66. doi: 10.1021/bi00050a029.
During epidermal growth factor mediated signal transduction, intracellular receptor autophosphorylation on tyrosine residues results in the localization of several SH2 domain bearing proteins, including c-src, to the plasma membrane. This process is part of a complex pathway of specific protein associations that culminates in the regulation of cell growth and mitogenesis. The SH2 domain-mediated interaction of c-src with the EGF receptor has been demonstrated, yet the precise function of c-src in EGF receptor signaling remains unclear. The phosphorylation of EGFR by c-src was studied in order to evaluate the molecular basis for this interaction. The C-terminal autophosphorylation domain of EGFR was extensively phosphorylated by c-src and EGFR kinase activities in vitro as determined by electrospay ionization mass spectrometry. The sites of phosphorylation within the autophosphorylation domain (residues 976-1186) were identified by LC/MS, LC/MS/MS, and Edman sequencing. The majority of the sites identified corresponded to the known autophosphorylation sites of EGFR. Kinetic analyses of site-specific phosphorylation were made combining very fast enzyme digests (< = or 2 min) and high-speed, perfusion chromatography. These studies revealed that Y1086 was phosphorylated to a significantly higher extent by c-src than by EGFR. Additionally, Y1101 was identified as a unique c-src phosphorylation site. The function of these phosphorylation sites with respect SH2 domain interactions was investigated by affinity chromatography/mass spectrometry. A subset of peptides corresponding to the eight possible tyrosine phosphorylation sites within the EGFR autophosphorylation domain was demonstrated to bind to the SH2 domain of c-src. Those which bound to the SH2 domain included peptides derived from EGFR sequences flanking Y992, Y1086, Y1101, and Y1148. These data indicate that specific EGF receptor c-src phosphorylation sites are also ligands for the SH2 domain of c-src. Finally, extensive c-src phosphorylation of EGFR promoted its conversion to a form that exhibits high-affinity (KD = 380 nM) and cooperative (Hill coefficient; n = 2) binding to the SH2 domain of c-src as measured by surface plasmon resonance. The identification of c-src phosphorylation sequences on EGFR as c-src SH2 binding sites supports the notion that this interaction plays a significant role in the regulation of growth factor receptor function and signal transduction.
在表皮生长因子介导的信号转导过程中，细胞内受体酪氨酸残基的自磷酸化导致包括c-src在内的几种含有SH2结构域的蛋白质定位于质膜。这一过程是特定蛋白质相互作用复杂途径的一部分，最终导致细胞生长和有丝分裂的调控。c-src与表皮生长因子受体之间由SH2结构域介导的相互作用已得到证实，但c-src在表皮生长因子受体信号传导中的精确功能仍不清楚。为了评估这种相互作用的分子基础，研究了c-src对表皮生长因子受体的磷酸化作用。通过电喷雾电离质谱法测定，在体外，表皮生长因子受体的C末端自磷酸化结构域被c-src和表皮生长因子受体激酶活性广泛磷酸化。通过液相色谱/质谱、液相色谱/串联质谱和埃德曼测序确定了自磷酸化结构域（第976 - 1186位氨基酸残基）内的磷酸化位点。所鉴定的大多数位点与表皮生长因子受体已知的自磷酸化位点相对应。结合非常快速的酶消化（<=或2分钟）和高速灌注色谱法对位点特异性磷酸化进行了动力学分析。这些研究表明，与表皮生长因子受体相比，c-src使Y1086的磷酸化程度显著更高。此外，Y1101被鉴定为一个独特的c-src磷酸化位点。通过亲和色谱/质谱法研究了这些磷酸化位点在SH2结构域相互作用方面的功能。已证明对应于表皮生长因子受体自磷酸化结构域内八个可能酪氨酸磷酸化位点的一部分肽段能与c-src的SH2结构域结合。那些与SH2结构域结合的肽段包括源自Y992、Y1086、Y1101和Y1148侧翼的表皮生长因子受体序列的肽段。这些数据表明，表皮生长因子受体特定的c-src磷酸化位点也是c-src的SH2结构域的配体。最后，通过表面等离子体共振测量，表皮生长因子受体广泛的c-src磷酸化促进其转变为一种对c-src的SH2结构域表现出高亲和力（解离常数KD = 380 nM）和协同性（希尔系数；n = 2）结合的形式。表皮生长因子受体上c-src磷酸化序列作为c-src SH2结合位点的鉴定支持了这种相互作用在生长因子受体功能调控和信号转导中起重要作用的观点。
