Gamper M, Howard P K, Hunter T, Firtel R A
Department of Biology, Center for Molecular Genetics, University of California, San Diego, 92093-0634, USA.
Mol Cell Biol. 1996 May;16(5):2431-44. doi: 10.1128/MCB.16.5.2431.
PTP3, the third nonreceptor protein tyrosine phosphatase identified in Dictyostelium discoideum, has a single catalytic protein tyrosine phosphatase domain. Recombinant PTP3 exhibited phosphatase activity that was inhibited by vanadate. PTP3 is expressed at a moderate level during growth. The level of transcripts increased between growth and 8 h of development and declined thereafter. Expression of lacZ under the control of the PTP3 promoter indicated a spatial localization of PTP3 in the anterior-like and prestalk cell types. There are two copies of the PTP3 gene in this haploid organism. Disruption of one copy led to a slow-growth phenotype. We were unable to obtain a strain with disruptions in both PTP3 genes. Overexpression of wild-type PTP3 led to slower growth rates and the formation of large aggregation streams. These streams split into smaller aggregates, many of which then arrested in development. Overexpression of a catalytically inactive mutation (Cys to Ser) had no effect on growth rate; however, this strain also formed large aggregation streams that later split up into large and small mound structures and became fruiting bodies of various sizes. Antiphosphotyrosine Western blot (immunoblot) analysis of total cell proteins showed that the pattern of protein tyrosine phosphorylation was specifically altered in PTP3 mutants. Addition of growth medium to starving cells and a subsequent replacement with nonnutrient buffer led to reciprocal changes in the pattern of several phosphotyrosine proteins, including a protein of approximately 130 kDa. Analysis of strains overexpressing active or inactive PTP3 suggested that p130 is a potential substrate of PTP3. A transient posttranslational phosphorylation of PTP3 further supported the role of PTP3 in these processes. The data obtained strongly suggest new regulatory functions for PTP3 that are distinct from those described earlier for D. discoideum PTP1 and PTP2.
PTP3是在盘基网柄菌中鉴定出的第三种非受体蛋白酪氨酸磷酸酶，具有单个催化性蛋白酪氨酸磷酸酶结构域。重组PTP3表现出被钒酸盐抑制的磷酸酶活性。PTP3在生长期间以中等水平表达。转录本水平在生长和发育8小时之间增加，此后下降。在PTP3启动子控制下的lacZ表达表明PTP3在前样细胞和柄细胞类型中呈空间定位。在这种单倍体生物中有两个PTP3基因拷贝。破坏一个拷贝导致生长缓慢的表型。我们无法获得两个PTP3基因均被破坏的菌株。野生型PTP3的过表达导致生长速率减慢并形成大的聚集流。这些流分裂成较小的聚集体，其中许多随后在发育中停滞。催化失活突变（半胱氨酸突变为丝氨酸）的过表达对生长速率没有影响；然而，该菌株也形成了大的聚集流，随后分裂成大小不一的丘状结构并变成各种大小的子实体。对总细胞蛋白进行的抗磷酸酪氨酸蛋白质印迹（免疫印迹）分析表明，PTP3突变体中蛋白质酪氨酸磷酸化模式发生了特异性改变。向饥饿细胞中添加生长培养基，随后用无营养缓冲液替换，导致几种磷酸酪氨酸蛋白的模式发生相反变化，包括一种约130 kDa的蛋白质。对过表达活性或无活性PTP3的菌株进行分析表明，p130是PTP3的潜在底物。PTP3的瞬时翻译后磷酸化进一步支持了PTP3在这些过程中的作用。所获得的数据强烈表明PTP3具有新的调节功能，这些功能不同于先前针对盘基网柄菌PTP1和PTP2所描述的功能。
