Regulatory Mechanisms of Microtubule Functions by MAP Kinase Cascade

我们分离了一个半显性拟南芥丙基酰胺过敏1-1 （PHS1 -1）突变体，该突变体对微管解聚药物丙基酰胺过敏，并证明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的磷酸酶在n端保守的mapk相互作用域发生氨基酸交换，保持了磷酸酶活性，并表现为显性负性。缺乏PHS1蛋白表达的几个PHS1 T-DNA插入空等位基因生长正常，未表现出明显的表型异常。我们分离出了PHS1 -1的多个基因抑制子，发现了PHS1的7个基因内抑制等位基因，进一步证明纯合的PHS1零等位基因不表现出明显的表型。其他磷酸酶可能有多余的功能。抑制因子筛选也恢复了几个基因外抑制突变体。其中一些基因外抑制等位基因被发现在微管蛋白基因中有突变。对剩余的抑制突变负责的基因正在通过基于图谱的方法被克隆。将GUS报告基因插入基因组区PHS1蛋白的c端，分析了PHS基因在转基因拟南芥中的表达情况。在几乎所有细胞类型中都检测到基础GUS活性，但在快速伸长的细胞中观察到强表达。当GFP-PHS1在其自身的启动子和终止子下表达时，野生型PHS1在细胞质中均匀分布，而PHS1 -1版本的PHS1在细胞质中以点状模式定位。

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.