van der Geer P, Wiley S, Pawson T
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0601, USA.
Oncogene. 1999 May 20;18(20):3071-5. doi: 10.1038/sj.onc.1202879.
Shc and IRS-1 (and their relatives) are cytoplasmic docking proteins that possess phosphotyrosine-binding (PTB) domains, through which they bind specific activated receptor tyrosine kinases (RTK). The subsequent phosphorylation of Shc or IRS-1 creates binding sites for the SH2 domains of multiple signaling proteins, leading to the activation of intracellular biochemical pathways. The PTB domains of Shc and IRS-1 both recognize autophosphorylation sites in RTKs with the consensus sequence NPXpY, but show distinct abilities to bind stably to RTKs such as the TrkA nerve growth factor receptor and the insulin receptor. In vitro analysis has suggested that residues N-terminal to the NPXpY motif may determine the affinity with which phosphopeptide ligands are recognized by the Shc and IRS-1 PTB domains. Unlike IRS-1, the Shc PTB domain binds poorly to the insulin-receptor (IR) beta subunit in vitro, owing to its low affinity for the NPXpY autophosphorylation site at Tyr 960 of the IR. As a consequence, Shc does not bind stably to the activated IR in cells. We show that substitution of Ser 955, five residues N-terminal to the Tyr 960 autophosphorylation site (the -5 position), with Ile alters the target specificity of the IR such that it stably associates with Shc in insulin-stimulated cells. A triple substitution of the -5, -8 and -9 residues relative to Tyr 960 of the IR to the corresponding amino acids found in the Shc PTB domain binding site of TrkA results in even stronger binding of the IR to Shc in vivo. The variant IRs with enhanced ability to bind Shc showed an increased ability to activate the MAPK pathway in response to insulin stimulation. These results demonstrate that subtle differences in residues N-terminal to NPXpY autophosphorylation sites determine the ability of RTKs to bind specific PTB domain proteins in vivo, and thus modify the signaling properties of activated receptors.
Shc和胰岛素受体底物-1（IRS-1，及其相关蛋白）是细胞质对接蛋白，它们具有磷酸酪氨酸结合（PTB）结构域，通过该结构域可结合特定的活化受体酪氨酸激酶（RTK）。Shc或IRS-1随后的磷酸化作用为多种信号蛋白的SH2结构域创造了结合位点，从而导致细胞内生化途径的激活。Shc和IRS-1的PTB结构域均识别具有共有序列NPXpY的RTK中的自磷酸化位点，但在与诸如TrkA神经生长因子受体和胰岛素受体等RTK稳定结合的能力上表现出明显差异。体外分析表明，NPXpY基序N端的残基可能决定Shc和IRS-1的PTB结构域识别磷酸肽配体的亲和力。与IRS-1不同，Shc的PTB结构域在体外与胰岛素受体（IR）的β亚基结合不佳，这是因为它对IR的Tyr 960处的NPXpY自磷酸化位点亲和力较低。因此，Shc在细胞中不会与活化的IR稳定结合。我们发现，将Tyr 960自磷酸化位点（-5位）N端的5个残基Ser 955替换为Ile，会改变IR的靶标特异性，使其在胰岛素刺激的细胞中与Shc稳定结合。将IR的Tyr 960相对的-5、-8和-9位残基替换为TrkA的Shc PTB结构域结合位点中发现的相应氨基酸，会使IR在体内与Shc的结合更强。与Shc结合能力增强的变异IR显示出在胰岛素刺激下激活丝裂原活化蛋白激酶（MAPK）途径的能力增强。这些结果表明，NPXpY自磷酸化位点N端残基的细微差异决定了RTK在体内结合特定PTB结构域蛋白的能力，从而改变了活化受体的信号传导特性。
