Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase

Abstract

Protein phosphatase 1 (PP-1) is known to be a critical component of eukaryotic cell cycle progression. In vitro, our previous studies showed that cdc2 kinase phosphorylates Thr-320 (T320) in PP-1, and that this leads to inhibition of enzyme activity. To examine directly the phosphorylation of PP-1 in intact mammalian cells, an antibody has been prepared that specifically recognizes PP-1C␣ phosphorylated at T320. Cell synchronization studies revealed in a variety of cell types that T320 of PP-1 was phosphorylated to high levels only during early to mid-mitosis. The phosphorylation of T320 of PP-1 was reduced by the cyclin-dependent protein kinase inhibitor, olomoucine, and increased by the PP-1͞PP-2A inhibitor, calyculin A. Immunof luorescence microscopy using phospho-T320 antibody indicated that in NIH 3T3 cells the phosphorylation of PP-1 began to increase from basal levels in prophase and to peak at metaphase. Immunostaining indicated that phospho-PP-1 was localized exclusively to nonchromosomal regions. Furthermore, in cell fractionation studies of mitotic cells, phospho-PP-1 was detectable only in the soluble fraction. These observations suggest that phosphorylation by cdc2 kinase in early to mid-mitosis and inhibition of PP-1 activity is likely to contribute to the increased state of phosphorylation of proteins that is critical to the initiation of normal cell division.Protein phosphorylation is widely recognized as the major mechanism that controls cell cycle progression. A family of cyclin-dependent protein kinases (CDKs) have been identified and found to be enzymes critical for the initiation and completion of DNA replication and cell division from yeast to mammals (1-5). The activity of CDKs is modulated through phosphorylation of their catalytic subunits and by association with activating or inhibiting proteins. For example, mitotic transition is mediated by the cdc2 kinase͞cyclin B complex, and activated cdc2 kinase has been found to drive dramatic structural reorganizations in the nuclear envelope, spindle apparatus, and chromosomal DNA by phosphorylation of a variety of substrates including histone, Eg5, lamin, vimentin, and plectin (6-12).Evidence suggests that serine͞threonine protein phosphatases function as crucial regulators of cell proliferation (4, 13-15). In particular, protein phosphatase 1 (PP-1), which is highly conserved in all eukaryotes, has been found to play a pivotal role in the cell cycle. Genetic studies have indicated that mutation of the enzyme in yeast, Aspergillus, and in Drosophila leads to a variety of defects in mitosis (16-18). Microinjection of anti-PP-1 antibody into B cells before cell division arrests cells at metaphase, whereas injection of PP-1 into anaphase cells accelerates cytokinesis (19). Expression of inhibitor-2, a specific PP-1 inhibitor, changes during the cell cycle, peaking during S phase and mitosis (20). Thus, the tightly balanced activity of CDKs and PP-1 appears to be required for normal cell division. However, the substrates that are targets f...

Roles of aurora‐A kinase in mitotic entry and G2 checkpoint in mammalian cells

“…In other words, signals that inactivate PP1 can promote the phosphorylation of aurora-A, consequently activating the aurora-A kinase. It was reported previously that PP1 is phosphorylated at threonine 320 by Cdc2 and is inactivated during early to mid-mitosis (Kwon et al 1997). Such evidence, together with our findings, led us to propose a model in which aurora-A kinase is activated by Cdc2 through the inhibition of PP1.…”

A model for restriction point control of the mammalian cell cycle

“…Dephosphorylation of Rb is catalysed by a type-1 protein phosphatase (PP1), whose activity is inhibited by cyclin/Cdk complexes (Dohadwala et al, 1994;Kwon et al, 1997 …”

Downregulation of Fer induces PP1 activation and cell-cycle arrest in malignant cells

“…The balance between these two opposing regulatory mechanisms determines the phosphorylation and activation state of pRB (Berndt et al, 1997). Phosphorylation of Thr320 in PP1a suppresses the phosphatase activity of that enzyme and increased phosphorylation level of Thr320 is inversely correlated with the phosphatase activity of PP1a in vivo (Dohadwala et al, 1994;Kwon et al, 1997;Liu et al, 1999). Conversely, dephosphorylation of the Thr320 site induces the phosphatase activity of PP1a toward pRB -siRNA or with fersiRNA in the absence or presence of cycloheximide.…”