Lu K P, Osmani S A, Osmani A H, Means A R
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
J Cell Biol. 1993 May;121(3):621-30. doi: 10.1083/jcb.121.3.621.
nimT encodes a protein in Aspergillus nidulans that is required for tyrosine dephosphorylation of p34cdc2 and has a strong homology to cdc25-type proteins. Conditional mutation of nimT (nimT23 mutation) arrests cells in G2 at the restrictive temperature. After release of the temperature-sensitive nimT23 block, p34cdc2 undergoes tyrosine dephosphorylation and we showed that as cells entered mitosis, a rapid increase in calmodulin was observed. The increase in calmodulin and progression into mitosis were prevented by reducing extracellular Ca2+ levels to 2 nM. The calmodulin gene of a nimT23-containing strain was replaced with a hybrid gene in which calmodulin transcription was regulated by the alcA promoter (AlcCaM/T23). This allowed experimental manipulation of the level of intracellular calmodulin by the carbon source in the medium. When either extracellular Ca2+ or intracellular calmodulin levels were reduced at the nimT23 G2 arrest point, p34cdc2 remained tyrosine phosphorylated but the mitotic NIMA kinase encoded by nimA was not activated. Release of the temperature sensitive nimT23 arrest when either extracellular Ca2+ or calmodulin concentrations were low blocked tyrosine dephosphorylation of p34cdc2, activation of NIMA and progression of cells into mitosis. However, reduced levels of either Ca2+ or calmodulin had no effect on the increase in histone H1 kinase activity associated with p13 beads or the degree of phosphorylation of the majority of MPM-2-reacting proteins following release of the nimT23 mutation. These results demonstrate that both Ca2+ and calmodulin are important for progression into mitosis from the nimT23 arrest point in a pathway involving activation of both NIMA and p34cdc2 protein kinases.
nimT在构巢曲霉中编码一种蛋白质，该蛋白质是p34cdc2酪氨酸去磷酸化所必需的，并且与cdc25型蛋白质具有高度同源性。nimT的条件性突变（nimT23突变）在限制温度下使细胞停滞在G2期。温度敏感的nimT23阻滞解除后，p34cdc2发生酪氨酸去磷酸化，并且我们发现当细胞进入有丝分裂时，观察到钙调蛋白迅速增加。通过将细胞外Ca2+水平降低到2 nM可阻止钙调蛋白的增加以及进入有丝分裂。将含有nimT23的菌株的钙调蛋白基因替换为一个杂合基因，其中钙调蛋白的转录由alcA启动子调控（AlcCaM/T23）。这使得能够通过培养基中的碳源对细胞内钙调蛋白水平进行实验性操作。当在nimT23 G2停滞点降低细胞外Ca2+或细胞内钙调蛋白水平时，p34cdc2仍保持酪氨酸磷酸化，但由nimA编码的有丝分裂NIMA激酶未被激活。当细胞外Ca2+或钙调蛋白浓度较低时，温度敏感的nimT23停滞的解除会阻止p34cdc2的酪氨酸去磷酸化、NIMA的激活以及细胞进入有丝分裂。然而，Ca2+或钙调蛋白水平的降低对与p13珠相关的组蛋白H1激酶活性的增加或nimT23突变解除后大多数MPM - 2反应蛋白的磷酸化程度没有影响。这些结果表明，在涉及NIMA和p34cdc2蛋白激酶激活的途径中，Ca2+和钙调蛋白对于从nimT23停滞点进入有丝分裂都是重要的。
