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.

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.

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.

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.

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.