Weinmaster G, Hinze E, Pawson T
J Virol. 1983 Apr;46(1):29-41. doi: 10.1128/JVI.46.1.29-41.1983.
The phosphorylation sites of the P140gag-fps gene product of Fujinami avian sarcoma virus have been identified and localized to different regions of this transforming protein. FSV P140gag-fps isolated from transformed cells is phosphorylated on at least three distinct tyrosine residues and one serine residue, in addition to minor phosphorylation sites shared with Pr76gag. Partial proteolysis with virion protease p15 or with Staphylococcus aureus V8 protease has been used to generate defined peptide fragments of P140gag-fps and thus to map its phosphorylation sites. The amino-terminal gag-encoded region of P140gag-fps contains a phosphotyrosine residue in addition to normal gag phosphorylation sites. The two major phosphotyrosine residues and the major phosphorserine residue are located in the carboxy-terminal portion of the fps-encoded region of P140gag-fps. P140gag-fps radiolabeled in vitro in an immune complex kinase reaction is phosphorylated at only one of the two C-terminal tyrosine residues phosphorylated in vivo and weakly phosphorylated at the gag-encoded tyrosine and at a tyrosine site not detectably phosphorylated in vivo. Thus, the in vitro tyrosine phosphorylation of P140gag-fps is distinct from that seen in the transformed cell. A comparative tryptic phosphopeptide analysis of the gag-fps proteins of three Fujinami avian sarcoma virus variants showed that the phosphotyrosine-containing peptides are invariant, and this high degree of sequence conservation suggests that these sites are functionally important or lie within important regions. The P105gag-fps transforming protein of PRCII avian sarcoma virus lacks one of the C-terminal phosphotyrosine sites found in Fujinami avian sarcoma virus P140gag-fps. Partial trypsin cleavage of FSV P140gag-fps immunoprecipitated with anti-gag serum releases C-terminal fragments of 45K and 29K from the immune complex that retain an associated tyrosine-specific protein kinase activity. This observation, and the localization of the major P140gag-fps phosphorylation sites to the C-terminal fps region, indicate that the kinase domain of P140gag-fps is located at its C terminus. The phosphorylation of P140gag-fps itself is complex, suggesting that it may itself interact with several protein kinases in the transformed cell.
已鉴定出藤浪禽肉瘤病毒P140gag-fps基因产物的磷酸化位点，并将其定位到这种转化蛋白的不同区域。从转化细胞中分离出的FSV P140gag-fps在至少三个不同的酪氨酸残基和一个丝氨酸残基上发生磷酸化，此外还有与Pr76gag共有的次要磷酸化位点。用病毒蛋白酶p15或金黄色葡萄球菌V8蛋白酶进行部分蛋白酶解，以产生P140gag-fps的特定肽片段，从而确定其磷酸化位点。P140gag-fps的氨基末端gag编码区域除了正常的gag磷酸化位点外，还含有一个磷酸酪氨酸残基。两个主要的磷酸酪氨酸残基和主要的磷酸丝氨酸残基位于P140gag-fps的fps编码区域的羧基末端部分。在免疫复合物激酶反应中体外放射性标记的P140gag-fps仅在体内磷酸化的两个C末端酪氨酸残基之一上发生磷酸化，在gag编码的酪氨酸和体内未检测到磷酸化的酪氨酸位点上磷酸化较弱。因此，P140gag-fps的体外酪氨酸磷酸化与转化细胞中的不同。对三种藤浪禽肉瘤病毒变体的gag-fps蛋白进行的胰蛋白酶磷酸肽比较分析表明，含磷酸酪氨酸的肽是不变的，这种高度的序列保守性表明这些位点在功能上很重要或位于重要区域内。PRCII禽肉瘤病毒的P105gag-fps转化蛋白缺少藤浪禽肉瘤病毒P140gag-fps中发现的一个C末端磷酸酪氨酸位点。用抗gag血清免疫沉淀的FSV P140gag-fps经部分胰蛋白酶裂解后，从免疫复合物中释放出45K和29K的C末端片段，这些片段保留了相关的酪氨酸特异性蛋白激酶活性。这一观察结果以及P140gag-fps主要磷酸化位点定位于C末端fps区域，表明P140gag-fps激酶结构域位于其C末端。P140gag-fps自身的磷酸化很复杂，这表明它可能在转化细胞中与几种蛋白激酶相互作用。
