Estrogens induce cell proliferation in target tissues by stimulating progression through G₁ phase of the cell cycle, but the underlying molecular targets remain undefined. To determine the role of the cyclin/cyclin-dependent kinase (CDK)/retinoblastoma protein (pRB) pathway in this response we treated MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 to inhibit estrogen-induced gene expression and induce G₁ phase arrest. Subsequent treatment with 17β-estradiol resulted in the synchronous entry of cells into S phase commencing at 12 h. The proportion of cells in S phase reached a maximum of 60% at 21–24 h. Cells subsequently completed mitosis and entered a second semisynchronous round of replication. Entry into S phase was preceded by increased activity of both Cdk4 and cyclin E-Cdk2 and hyperphosphorylation of pRB, all within the first 3–6 h of estradiol treatment. The increase in Cdk4 activity was accompanied by increases in cyclin D1 mRNA and protein, indicating that an initiating event in the activation of Cdk4 was increased cyclin D1 gene expression. In contrast, the levels of Cdk2 and the CDK inhibitors p21 (WAF1/CIP1/SDI1) and p27 (KIP1) in total cell lysates and in cyclin E immunoprecipitates were unaltered at these early time points. However, an inhibitory activity was present in antiestrogen-pretreated cell lysates toward recombinant cyclin E-Cdk2 and was relieved by estradiol treatment. This activity was attributable predominantly to p21. These apparently conflicting data were resolved by performing gel filtration chromatography, which revealed that only a minority of cyclin E-Cdk2 complexes were active following estradiol treatment. Active complexes eluted at a higher molecular weight than inactive complexes, were relatively deficient in both p21 and p27, and contained Cdk2 with increased threonine 160 phosphorylation, consistent with a mechanism of activation of cyclin E-Cdk2 involving both reduced CDK inhibitor association and CDK-activating kinase-mediated phosphorylation of Cdk2. These results provide an explanation for the early activation of both cyclin D1-Cdk4 and cyclin E-Cdk2 complexes that accompany G₁-S phase progression in response to estradiol.

Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin‐depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi‐protein complex consisting of SSH‐1L, LIMK1, actin, and the scaffolding protein, 14‐3‐3ζ, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH‐1L interact in vitro and co‐localize in vivo , and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH‐1L is F‐actin dependent and is negatively regulated via phosphorylation by PAK4. 14‐3‐3ζ binds to phosphorylated slingshot, decreases the amount of slingshot that co‐sediments with F‐actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.

The natural arrest of vertebrate unfertilized eggs in second meiotic metaphase results from the activity of cytostatic factor (CSF). The product of the c-mos(xe) proto-oncogene is thought to be a component of CSF and can induce metaphase arrest when injected into blastomeres of two-cell embryos. The c-Mos(xe) protein can directly activate the mitogen-activated protein kinase kinase (MAP kinase kinase) in vitro, leading to activation of MAP kinase. MAP kinase and c-Mos(xe) are active in unfertilized eggs and are rapidly inactivated after fertilization. Microinjection of thiophosphorylated MAP kinase into one blastomere of a two-cell embryo induced metaphase arrest similar to that induced by c-Mos(xe). However, only arrest with c-Mos(xe) was associated with activation of endogenous MAP kinase. These results indicate that active MAP kinase is a component of CSF in Xenopus and suggest that the CSF activity of c-Mos(xe) is mediated by MAP kinase.