Regulatory Mechanisms of Microtubule Functions by MAP Kinase Cascade

我们分离到了一个对微管解聚药物丙氨酰胺表现出超敏反应的半显性拟南芥突变体丙氨酰胺1-1(phs1-1)，并证明了PHS1编码一种属于有丝分裂原激活的新蛋白。蛋白激酶(MAPK)磷酸酶家族。Phs1-1中的磷酸酶在N端保守的MAPK相互作用区域进行了氨基酸交换，保持了磷酸酶的活性，表现为显性负作用；PHS1中几个缺乏PHS1蛋白表达的T-DNA插入缺失等位基因生长正常，没有表现出任何明显的表型异常。我们分离了许多phs1-1的基因抑制基因，发现了PHS1的7个基因内抑制等位基因，进一步证明纯合子PHS1缺失等位基因没有明显的表型。其他的磷酸酶可能有多余的功能。抑制子筛选还发现了几个基因外抑制子突变体。其中一些基因外抑制等位基因被发现在微管蛋白基因上发生了突变。其余抑制突变体的相关基因正在通过作图的方法进行克隆。通过将GUS报告基因插入到基因组区域PHS1蛋白的C末端，分析了PHS基因在转基因拟南芥中的表达。基本的GUS活性在几乎所有类型的细胞中都可检测到，但在快速伸长的细胞中观察到强烈的表达。当GFP-PHS1在其各自的启动子和终止子作用下表达时，野生型PHS1在胞浆中均匀分布，而phs1-1在细胞质中呈点状分布。

We isolated a semi-dominant Arabidopsis thaliana mutant propyzamide hypersensitive 1-1 (phs1-1) which showed hypersensitivity toward a microtubule-depolymerizing drug, propyzamide, and demonstrated that PHS1 codes for a novel protein belonging to a mitogen-activated. protein kinase (MAPK) phosphatase family. The phosphatase in phs1-1 had an amino acid exchange at the conserved MAPK-interaction domain at the N-terminus, retained a phosphatase activity, and acted dominant negatively.Several T-DNA insertion null alleles of PHS1 which lacked expression of the PHS1 protein grew normally and did not show any obvious phenotypic abnormalities. We isolated many genetic suppressors of phs1-1, and found 7 intragenic suppressor alleles of PHS1, further demonstrating that homozygous PHS1 null alleles do not show obvious phenotypes. Other phosphatases may have redundant functions. The suppressor screening also recovered several extragenic suppressor mutants. Some of these extragenic suppressor alleles were found to have mutations in tubulin genes. Responsible genes for the remaining suppressor mutants are being cloned by a map-based approach.Expression of the PHS gene was analyzed in transgenic Arabidopsis plants in which a GUS reporter gene was inserted in the C-terminus of the PHS1 protein in the genomic region. Basal GUS activities were detected in almost all cell types but the strong expression was observed in rapidly elongating cells. When GFP-PHS1 was expressed under its own promoter and terminator, wild-type PHS1 was found uniformly in the cytosol whereas the phs1-1 version of PHS1 was localized in a dot-like pattern in the cytoplasm.