Attenuation of p38 MAPK activity upon contact inhibition in fibroblasts.

The molecular events, which govern growth control upon contact inhibition have not yet been clearly defined. Previous work has indicated that there is an increase in the expression of mitogen-activated protein kinase phosphatases (MKPs) upon the attainment of contact inhibition in normal fibroblasts, concurrently with a decrease in ERK activity. To investigate the potential role of p38 and JNK in the transition to a contact-inhibited state, normal human fibroblasts (BJ) were grown to subconfluent and confluent densities. The total levels and phosphorylation states of p38 and JNK were assayed, and were compared to protein levels seen in HT-1080 fibrosarcoma cells, which lack contact-inhibited growth control. Activation of JNK was not apparent in these cells, though p38 was found to be active in proliferating cells, but attenuated in contact-inhibited cultures. Such fluctuations in p38 activity were not seen in cultures of fibrosarcoma cells of increasing density. This alteration in p38 activity was also reflected by attenuated activation of the downstream transcription factor ATF-2 upon contact inhibition. Overexpression of MKP-1 in fibrosarcoma cells and fibroblasts reduced proliferation, while expression of a phosphatase-resistant p38 protein (p38(N316)) enhanced proliferation of normal fibroblasts. Taken together, these results suggest the involvement of negative regulation of p38 in contact-inhibited growth control.

ERK regulation upon contact inhibition in fibroblasts.

Despite the understanding of the importance of mitogen-activated protein (MAP) kinase activation in the stimulation of growth, little is known about the role of MAP kinase regulation during contact inhibited growth control. To investigate the role of the MAP kinase extracellular signal-regulated kinase (ERK) during the transition to a contact inhibited state, cultures of normal fibroblasts (BJ) were grown to different stages of confluency. The levels of MAP kinase phosphatase (MKP) expression and the amount of active ERK and MAP ERK kinase (MEK) in these cultures were assessed through western blot analysis and were compared to fibrosarcoma cell cultures (HT-1080), which lack contact inhibition. In normal fibroblasts, the amounts of active MEK and ERK decline at contact inhibition, concurrently with a rise in MKP-1, MKP-2, and MKP-3 protein levels. In contrast, fibrosarcoma cells appear to lack density-dependent regulation of the ERK pathway. Additionally, altering the redox environment of fibrosarcoma cells to a less reducing state, as seen during contact inhibition, results in increased MKP-1 expression. Taken together, these results suggest that the altered redox environment upon contact inhibition may contribute to the regulation of ERK inactivation by MKPs.

Cathepsin L plays an active role in involution of the mouse mammary gland.

Involution of the mammary gland after weaning occurs in two stages. The first stage is reversible, whereas the second stage is characterized by the irreversible collapse of the alveolar structure. A differential display analysis using cDNAs from tissues obtained at various times after forced weaning of pups identified cathepsin L as up-regulated during early involution. Levels of cathepsin L mRNA were dramatically increased within 24 hr after weaning. Cathepsin L protein detected by immunoblot was also increased during involution, reaching near maximal levels by 36 hr after weaning. In situ immunohistochemistry detected pronounced cathepsin L protein in the cytoplasm and cell periphery. Mice treated with a specific inhibitor of cathepsin L exhibited substantially reduced numbers of apoptotic cells at times up to 72 hr after weaning when compared with untreated animals. The cathepsin L inhibitor did not alter levels of cathepsin L detected in immunoblots or influence molecular weight of the cathepsin L species detected. These data suggest that cathepsin L plays a regulatory role early in the process of mammary gland involution.

The carboxyl-terminal domains of MKP-1 and MKP-2 have inhibitory effects on their phosphatase activity.

Both the mitogen-activated protein kinase (MAPK) phosphatases MKP-1 and MKP-2 exert important feedback control of MAPK-mediated signaling events. The function of MKP-1 and MKP-2 is regulated via complex mechanisms, ranging from increased transcription of the MKP-1 and MKP-2 genes to post-translational catalytic activation of MKP-1 and MKP-2 proteins upon binding to their substrate MAPKs. In addition, MKP-1 stability increases upon ERK-dependent phosphorylation of two serine residues in its C-terminus. The C-terminal regions of MKP-1 and MKP-2, but not those of other MKPs, are homologous. To investigate the role of this domain, we have deleted the C-terminal tails from MKP-1 and MKP-2 and examined the effect of these deletions on their enzymatic activity. C-terminally truncated MKP-1 and MKP-2 exhibited, both in vivo and in vitro, substantially greater phosphatase activity towards their substrate MAPKs than did the full-length counterparts. However, C-terminal truncations did not significantly change either their substrate affinity, or their substrate-mediated catalytic activation. Basal phosphatase activity of the truncated proteins was also significantly higher than that of the wild-type counterparts. Collectively, these results suggest that the C-terminal domain may potentially play a role in the regulation of MKP-1 and MKP-2.

A mammalian expression system for rapid production and purification of active MAP kinase phosphatases.

Expression of enzymatically active mammalian proteins in Escherichia coli can proven to be a challenging task due to poor solubility, improper folding, and lack of adequate posttranslational modification. Expression of mammalian proteins using baculovirus or yeast systems is time-consuming and may also be subject to inadequate modification. In order to overcome these technical difficulties, we have developed a mammalian expression system for the convenient subcloning of cDNA fragments, high-level expression, and one-step purification of enzymatically active proteins. The mammalian expression vector pEBG that expresses glutathione S-transferase fusion proteins was modified to create an SrfI restriction site in the multiple cloning site. The protein coding sequences of MAP kinase phosphatase-1 (MKP-1), MAP kinase phosphatase-2 (MKP-2), and the tumor suppressor PTEN were PCR-amplified using Pfu DNA polymerase and cloned into the SrfI site through SrfI digestion-coupled ligation. The resulting plasmids were transiently transfected into 293T cells using FuGENE 6 transfection reagent. Forty eight hours after transfection, cells were harvested and bioactive recombinant proteins were purified by glutathione-Sepharose beads. Protein yield, which ranged from 200 to 700 microg, was more than adequate for biochemical studies. The usefulness of this versatile system for studying protein function and its potential application for proteomics research are discussed.