Regulatory Mechanisms of Microtubule Functions by MAP Kinase Cascade

我们分离出一个半显性拟南芥突变体propyzamide超敏1-1（phs 1 -1），它对微管解聚药物propyzamide表现出超敏反应，并证明PHS 1编码一种新的蛋白质，属于促分裂原激活。蛋白激酶（MAPK）磷酸酶家族。phs 1 -1中的磷酸酶在保守的MAPK相互作用结构域N端发生了氨基酸交换，保留了磷酸酶活性，并呈显性负作用，而几个T-DNA插入无效等位基因没有表达PHS 1蛋白，它们生长正常，没有表现出任何明显的表型异常。我们分离了许多phs 1 -1的基因抑制子，并发现了7个基因内抑制等位基因，进一步证明纯合的phs 1无效等位基因没有表现出明显的表型。其他磷酸酶可能具有冗余功能。抑制子筛选还回收了几个基因外抑制子突变体。发现这些基因外抑制等位基因中的一些在微管蛋白基因中具有突变。其余的抑制突变体的负责基因正在通过基于图位的approach. PHS基因的表达进行了分析，在转基因拟南芥植物中，GUS报告基因插入在基因组区域的PHS 1蛋白的C-末端。在几乎所有的细胞类型中检测到基本GUS活性，但在快速伸长的细胞中观察到强表达。当GFP-PHS 1在其自身的启动子和终止子下表达时，发现野生型PHS 1均匀地存在于细胞质中，而phs 1 -1版本的PHS 1以点状图案定位于细胞质中。

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.