Oskam R, van de Meerendonk W P, de Boer A J, Wind J C, Rijksen G, Staal G E
Department of Hematology, Academic Hospital Utrecht, The Netherlands.
Tumour Biol. 1987;8(4):186-202. doi: 10.1159/000217522.
The kinetics of endogenous protein phosphorylation and resultant phosphoprotein patterns were investigated in well-differentiated (DMTC) and undifferentiated (AMTC) medullary thyroid carcinomas of the rat. Cytosolic or particulate fractions from these tumors were incubated with gamma-32P-ATP in the presence of various effectors. Phosphorylation appeared to be predominantly independent of exogenously added cyclic AMP. Magnesium and manganese were equally effective cofactors. For both tumor types 32P incorporation into cytosolic proteins was maximal within 3-4 min after addition of ATP and subsequently decreased gradually within 1 h. In contrast, in particulate preparations maximal incorporation was reached within 30 s and remained constant over a relatively long time span. In both cases, however, 32P incorporation in extracts from DMTCs were 50-100% higher as compared to AMTCs. Comparison of the phosphoprotein patterns of each tumor after in vitro phosphorylation showed some significant differences. A phosphoprotein with molecular weight of 90 kilodalton (90 kD) was exclusively expressed in the cytosol of DMTC, whereas 99- and 84-kD phosphoproteins were only present in the cytosol of AMTC. The DMTC particulate fraction contained two phosphoproteins (with molecular weights of 40 and 37 kD), which were absent from that of AMTC. In addition, a number of proteins were more intensely phosphorylated in one of the tumors, e.g. proteins of 94 and 33 kD in DMTC cytosol and a protein of 78 kD in AMTC cytosol. Calcium induced phosphorylation of five proteins in DMTC cytosol (with molecular weights of 69, 55, 49, 43 and 32 kD), which were less intensely or not phosphorylated in AMTC. Tyrosine kinase activity was investigated using exogenously added poly(glutamine:tyrosine, 4:1) as an artificial substrate. Cytosolic tyrosine kinase activity in DMTC was +/- 50% higher than in AMTC (11.9 +/- 0.6 and 8.2 +/- 1.7 pmol/mg/min, respectively). The enzyme activities in the particulate preparations were much higher than in the cytosols (+/- 100 pmol/mg/min), although considerable variations in enzyme activity between different tumors of either type were observed. Quantitative differences in tyrosine kinase activity between AMTC and DMTC particulate fractions did not seem to exist using this substrate. Phosphoamino acid analysis of endogenously phosphorylated proteins in both AMTC and DMTC showed phosphotyrosine to be present only in cytosolic proteins within the 50-kD molecular weight region, the majority of 32P being on serine and some on threonine.(ABSTRACT TRUNCATED AT 400 WORDS)
研究了大鼠高分化（DMTC）和未分化（AMTC）甲状腺髓样癌中内源性蛋白质磷酸化动力学及由此产生的磷蛋白模式。将这些肿瘤的胞质或颗粒部分与γ-32P-ATP在各种效应物存在下孵育。磷酸化似乎主要独立于外源添加的环磷酸腺苷。镁和锰是同样有效的辅助因子。对于两种肿瘤类型，加入ATP后3 - 4分钟内，32P掺入胞质蛋白的量达到最大，随后在1小时内逐渐下降。相比之下，在颗粒制剂中，最大掺入在30秒内达到，并在相对较长的时间内保持恒定。然而，在这两种情况下，DMTC提取物中的32P掺入量比AMTC高50 - 100%。体外磷酸化后每种肿瘤的磷蛋白模式比较显示出一些显著差异。一种分子量为90千道尔顿（90 kD）的磷蛋白仅在DMTC的胞质中表达，而99 kD和84 kD的磷蛋白仅存在于AMTC的胞质中。DMTC颗粒部分含有两种磷蛋白（分子量分别为40和37 kD），而AMTC颗粒部分没有。此外，一些蛋白质在其中一种肿瘤中磷酸化程度更高，例如DMTC胞质中的94 kD和33 kD蛋白质以及AMTC胞质中的78 kD蛋白质。钙诱导DMTC胞质中五种蛋白质（分子量分别为69、55、49、43和32 kD）磷酸化，而这些蛋白质在AMTC中磷酸化程度较低或未磷酸化。使用外源添加的聚（谷氨酰胺：酪氨酸，4:1）作为人工底物研究酪氨酸激酶活性。DMTC中的胞质酪氨酸激酶活性比AMTC高约50%（分别为11.9±0.6和8.2±1.7 pmol/mg/min）。颗粒制剂中的酶活性比胞质中的高得多（约100 pmol/mg/min），尽管在两种类型的不同肿瘤之间观察到酶活性有相当大的差异。使用这种底物时，AMTC和DMTC颗粒部分之间酪氨酸激酶活性的定量差异似乎不存在。对AMTC和DMTC中内源性磷酸化蛋白质的磷酸氨基酸分析表明，磷酸酪氨酸仅存在于分子量50 kD区域内的胞质蛋白中，大部分32P位于丝氨酸上，一些位于苏氨酸上。（摘要截断于400字）
