第二节 细胞周期的调控• 一、 MPF 的发现和作用 Rao 和

Johnson(1970 、 1972 、 1974) 将 Hela细胞同步于不同阶段，然后与 M 期细胞混合，在灭活仙台病毒介导下，诱导细胞融合，发现与 M 期细胞融合的间期细胞产生了形态各异的早熟凝集染色体(prematurely condensed chromosome ， PCC) ，这种现象叫做早熟染色体凝集 (premature chromosome condensation) 。

** MPF, the mitosis-promoting factor

Rao,70’

PCC 早熟染色体凝集G1+M

S+M

G2+M

PCC 现象说明 M 期细胞中有可能存在一种诱导染色体凝集的因子。

S 期激活因子简称 SPF

的细胞融合实验1,S+G1 结果是 G1 早进入 S 期（和 S 期激活因子有关）2, G2+S 结果 S 期细胞继续合成 DNA ，说明 G2 期并未产生 DNA 合成抑制因子。但 G2 也不受 S 期诱导， G2 不合成 DNA ，说明在一个细胞周期中 DNA只合成一次。

3 ， G2+G1 结果 G2 不能诱导 G1 期合成 DNA 。

有人做如下提取液

G1 提取液G2 提取液S 提取液M 提取液

将这些提取液分别注射的蛙卵母细胞中，发现 G1 和 S 期不具 MPA （促成熟活性）MPA 在 G2-M 过度期达到高峰，这时正是染色体形成时期

有人将这种诱导减数分裂的物质称为有丝分裂因子（ MF ）后来称做 MPF （成熟促进因子）

* MPF, the maturation-promoting factor

Y. Masui and C. L. Markert. 1971. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes J. Exp. Zool. 177: 129-145.

• 不仅同类 M 期细胞可以诱导 PCC ，不同类的 M 期细胞也可以诱导 PCC 产生，如人和蟾蜍的细胞融合时同样有这种效果，这就意味着 M 期细胞具有某种促进间期细胞进行分裂的因子，即成熟促进因子 (maturation promoting factor ， MPF) 。

• 早在 1960s ， Yoshio Masui 发现成熟蛙卵的提取物能促进未成熟卵的胚胞破裂(Germinal Vesicle Breakdown ， GVBD) ，后来 Sunkara 将不同时期 Hela 细胞的提取液注射到蛙卵母细胞中，发现 G1 和 S 期的抽取物不能诱导 GVBD ，而 G2和 M 期的则具有促进胚胞破裂的功能，它将这种诱导物质称为有丝分裂因子 (MF) 。后来在 CHO 细胞，酵母和粘菌中也提取出相同性质的 MF 。这类物质被统称为 MPF 。

• 1960s Leland Hartwell 以芽殖酵母（图 13-16 ）为实验材料，利用阻断在不同细胞周期阶段的温度敏感突变株（在适宜的温度下和野生型一样），分离出了几十个与细胞分裂有关的基因 (cell division cycle gene ， CDC) 。如芽殖酵母的 cdc28 基因，在 G2/M 转换点发挥重要的功能。 Hartwell 还通过研究酵母菌细胞对放射线的感受性，提出了 checkpoint （细胞周期检验点）的概念，意指当 DNA 受到损伤时，细胞周期会停下来。

• 1970s Paul Nurse 等人以裂殖酵母（图）为实验材料，同样发现了许多细胞周期调控基因，如：裂殖酵母 cdc2 、 cdc25的突变型和在限制的温度下无法分裂；

wee1 突变型则提早分裂，而 cdc25 和wee1 都发生突变的个体却会正常地分裂（图）。

• 进一步的研究发现 cdc2 和 cdc28 都编码一个 34KD 的蛋白激酶，促进细胞周期的进行。而 weel 和 cdc25 分别表现为抑制和促进 CDC2 的活性。这也解释了为何 cdc25 和 wee1 双重突变的个体可以恢复野生型的表型。

二、 P34CDC2 的发现和细胞周期调控因子 MPF

的关系MPF 的主要成分就是 P34CDC2 和 P56CDC13 的同源物。

细胞周期蛋白为调节亚基P34CDC2 为催化亚基

正是由于不同的细胞周期蛋白和不同的 CDC2 基因产物的结合、分离、磷酸化和去磷酸化，激活催化亚基的活性，而成为驱动细胞周期运转的引擎。

The story of the work in identifying the key regulators of the cell division cycle and the 2001 Nobel Prize for Physiology or Medicine

Nurse is Director General of the Cancer Research UK ( was the ICRF) and has his research group.

In the mid-1970s, Nurse was working in the Scottish city of Edinburgh as a postdoc studying the fission yeast S. pombe. He found some mutant cells that were constantly small even though they multiplied at the same rate as other cells in the population.

A, Nurse found cdc2

The difference was that they appeared to divide before they had reached full size. He went on to isolate 50 of these mutants which he called wee ， meaning small in the Scottish dialect; 49 had mutations in a gene he called wee1, and one was mutated in a different gene, wee2.

• Nurse realised that wee2 is crucial for cell division when he noticed that cells can enter mitosis if this gene is mutated so that its protein kinase product loses its activity, whereas cells with hyperactive wee2 enter mitosis prematurely. The wee2 gene was later renamed cell division cycle 2 (CDC2).

In the cdc2- mutant, which has a recessive phenotype, the cdc2 gene is inactivated. At the restrictive condition, the cell fails to pass the mitotic entry checkpoint, continues growing, and so becomes enormously large.

C, the discovery of cdc mutants of fission yeast

Conversely, the dominant cdc2 mutant, cdc2D, in which the cdc2 gene product is hyperactive, passes the checkpoint prematurely, at an abnormally small size.

(Photographs by S.Moreno)p34cdc2, a serine/threonine protein kinase

In 1982, Nurse and his colleagues found that CDC2 was identical in function to CDC28, one of the hundred or so cell cycle-related genes that, father of the field, Hartwell had previously isolated from bakers yeast, S. cerevisiae. This was fitting as Nurse‘s work was originally inspired by Hartwell’s four seminal papers published in 1970 and 1971 on genetic control of the cell division cycle in yeast. Hartwell went on to characterise many of these S. cerevisiae cdc genes and also, with Ted Weinert, to develop the concept of checkpoints in the cell cycle that verify that the cell‘s DNA is intact and complete before allowing the division to procede.

Hartwell found cdc28

三、细胞周期蛋白1 P34CDC2+ 周期蛋白的引擎作用外还有若干个检验点的监视。2 P34CDC2 的量是相对恒定的，而细胞周期蛋白是随着细胞周期的变化而波动的。

周期蛋白和 P34CDC2 结合在一定条件下激活P34CDC2 的活性

Hunt was working on a totally unrelated project. His background was in protein synthesis and its control. "I was a rather odd creature of a sort of mixture of a biochemist and a molecular physiologist, and I was interested in seeing how systems worked," explained Hunt, who was looking at how sea urchin eggs turn on protein synthesis after they are fertilised.

Tim Hunt found cyclin in 1983

One day, Hunt did an experiment to look at the pattern of protein synthesis in fertilised eggs when they start to divide. "I have no real idea why I did the experiment ?it just seemed a good idea at the time," he admits. "I decided to take time points. When the gel was developed on Friday 23 July 1982, I found that the strongest band early on suddenly got very much weaker at about the time when the eggs were about to divide in two. This was a stunning, startling discovery."

Immediately, Hunt realised the implications. Researchers were aware that some stages of the cell cycle required protein synthesis, but until then nobody in the field had even remotely suggested that something would be specifically degraded to allow a cell to progress from one stage of the cell cycle to another. The disappearing protein became known as cyclin. "Twenty years later people are still struggling to find out how this mechanism works," says Hunt.

# Behavior of the temperature-sensitive cell-division-cycle (cdc) mutants

Most conditional cell-cycle mutants are temperature-sensitive mutants in which the mutant protein fails to function at high temperatures but functions well enough to allow cell division at low temperatures. A temperature-sensitive cdc mutant strain of cells can be grown at low temperature (the permissive condition) and then raised to a higher temperature (the restrictive condition) to switch off the function of the affected gene. All of the cells will continue their cycling until they reach the point where the function of the mutant gene is required for further progress, and at this point they will all stop

• 蛙 P32 + P45• 裂殖酵母 P34CDC2 + P56CDC13

• 海胆卵细胞 cdc2 蛋白 + 周期蛋白 B

CyclinsA2

B1

C

D

E

Cln3

F

G

Destruction BoxR42

PEST

PEST

PEST

PEST

PEST

48 kDa

48 kDa

33 kDa32 kDa

44 kDa

66 kDa87 kDa

29 kDa

6, the molecular features of Cyclins

CYCLIN BOX

细胞周期蛋白哺乳动物 -M 期有 周期蛋白 B

S 期有周期蛋白 A

酵母中 G1 期周期蛋白，有 CLN1

CLN2 ， CLN3

脊椎动物中有周期蛋白 C 、 D 、 E 等。

四、 CDK 激酶和它的抑制物 401

Cyclin-dependent kinase(CDK)

表 11-2 P401

可以看到 CDK 激酶的种类、可以结合的周期蛋白以及执行功能的可能时期。

Cdc2 (CDK1)

CDK2

CDK3

CDK4

CDK5

PV/STVRE

PSSALRE

CDK6PLSTIRE

CDK7 (p40MO15)NRTAIRE

PSTAIRE

PSTAIRE

PSTAIRE65%

66%

44%

57%47%

40%

100%

7, the molecular features of CDKs

五、细胞周期运转调控 402

（一） G2 和 M 期转化与 CDK1 激酶的调控作用

• 实际上 CDK 激酶和周期蛋白结合后仍不能激活激酶。还需要其他激酶如 CAK 激酶（ CDK1 活性激酶）。

b, by phosphorylatinon/dephospholylation of CDK subunit

Cyclin B/A Cyclin B/A

+

+ Cdc25c

CDK1

Thr14 Tyr15

Thr161

Cyclin B/A

CDK1

Thr14 Tyr15

Thr161

P P

P

Cyclin B/A

CDK1

Thr14 Tyr15

Thr161

APC +

P

+ CAK

Inactive

InactiveActive

21-10

• （二） M 期周期蛋白与分裂中期向后期转化

E1 泛素激活酶E2 泛素结合酶E3 泛素连接酶

• 1 APC 诱导 M 期周期蛋白泛素化。还调节非周期蛋白类的降解。• 2 细胞分裂走向后期的调控• Mad2 蛋白在动粒上和 Cdc20 结合，抑制

APC 活性，因为 Cdc20 是 APC 的正调控• 因子。 动粒被微管捕获后， Mad2 蛋白离开动粒并释放 Cdc20 ， Cdc20 激活

APC 。• APC 诱导周期蛋白泛素化使 CDK 激酶活性丧失。

浓度

蛙胚胎发育细胞分裂

Fluctuation of cyclin and MPF levels during the cell cycle.

A model for cell cycle regulation in fission yeast.

至少在 cell cycle of fission yeast. 可以看到两个控制点：START 和 G2-M 。

需要同样的 cdc2激酶和不同的cyclin 。fission yeast 有两组 cyclin ： G1 cyclin 和 mitotic cyclin.

Progression through the cell cycle requires the phosphorylation and dephosphorylation of critical cdc2 residues.

An example of the importance of subcellular localization in cell cycle requlation. 活 Hela cell 荧光标记 cyclin B1 被大量注射入细胞。 可以看到 G2 phase cyclin B1 几乎都在细胞质， same cell 在 prophase of mitosis 在细胞核积累。 cyclin B1是动物细胞的一个重要的细胞周期蛋白，它在 mitosis之前可以自由进出细胞核，到了prophase of mitosis 就会在细胞核积累，这是因为 cyclin B1 的核输出信号（ NES ）的一个氨基酸残基被磷酸化了。这样阻碍 cyclin B1向外运输即返回细胞质。如果没有这种cyclin B1 在细胞核的积累，细胞 mitosis 就不能开始。

Combinations between various cyclins and cyclin-dependent kinases at different stages in the mammalian cell cycle.

A model for the mechanism of action of a DNA-damage checkpoint.

The spindle checkpoint.