Coordination of Protein Kinase and Phosphoprotein Phosphatase Activities in Mitosis

Abstract

Dynamic changes in protein phosphorylation govern the transitions between different phases of the cell division cycle. A "tug of war" between highly conserved protein kinases and the family of phosphoprotein phosphatases (PPP) establishes the phosphorylation state of proteins, which controls their function. More than three-quarters of all proteins are phosphorylated at one or more sites in human cells, with the highest occupancy of phosphorylation sites seen in mitosis. Spatial and temporal regulation of opposing kinase and PPP activities is crucial for accurate execution of the mitotic program. The role of mitotic kinases has been the focus of many studies, while the contribution of PPPs was for a long time underappreciated and is just emerging. Misconceptions regarding the specificity and activity of protein phosphatases led to the belief that protein kinases are the primary determinants of mitotic regulation, leaving PPPs out of the limelight. Recent studies have shown that protein phosphatases are specific and selective enzymes, and that their activity is tightly regulated. In this review, we discuss the emerging roles of PPPs in mitosis and their regulation of and by mitotic kinases, as well as mechanisms that determine PPP substrate recognition and specificity.

Keywords: kinases and phosphatase; mass spectrometry; mitosis; phosphatases; protoemics.

Figure 1

Fine tuning between the mitotic protein kinases and protein phosphatases regulates mitotic progression. The relative activities of major mitotic protein kinases including Cdk1, AURKA, AURKB, and Plk1, indicated in blue spectrum, increase as the cells enter mitosis. This increase is accompanied by a relative decrease in the activities of major mitotic phosphatases including PP1, PP2A-B56 and PP2A-B55. While PP2A-B55 activity is completely inhibited by binding of its inhibitors ENSA and ARPP-19 at mitotic entry, PP2A-B56 is still active at localized complexes and regulates mitotic progression. Similarly, most of the PP1 activity is inhibited by Cdk1 dependent phosphorylation of its C-terminal Thr-320 residue at mitotic entry, but localized PP1 complexes remain active during mitosis. PP1 regains complete activity after the degradation of cyclin B and consequent decline of Cdk1 activity at metaphase-anaphase transition and controls the exit of cells from mitosis.

Figure 2

Protein kinases and protein phosphatases regulate each other during mitosis. Protein kinases and protein phosphatases coordinate with each other through underlying dynamic phosphorylation changes within kinase/phosphatase catalytic or regulatory subunits. Cdk1/cyclin B, directly or indirectly, inhibits the phosphatase activity of PP1, PP2A-B55, and PP4. Conversely, PP1 suppresses the kinase activity of AURKB through its regulatory protein Sds22 (PPP1R7), Plk1 through Mypt1 (PPP1R12A) and Gwl through PPP1R3B. Phosphorylation by Cdk1/cyclin B within or near the PP2A-B56 binding LxxIxE motif in substrates increases the affinity of PP2A-B56 interactions. PP2A-B55 activity is inhibited during mitosis by Gwl phosphorylation of the inhibitory proteins ENSA and ARPP-19. PP1 inactivates Gwl at mitotic exit, thereby activating PP2A-B55. PP6 is the T-loop phosphatase for AURKA, thereby decreasing its activity directly.

Figure 3

Modes of linear motif recognition by protein phosphatases. (A) Linear motifs can dictate the binding preferences of PPP family phosphatases. This can include a preference for the phosphosite (serine or threonine) or a preference for the residues surrounding the phosphosite (basic, acidic or proline-directed). (B) PPPs recognize short linear motifs (SLiMs) in regulatory proteins or substrates. PPP binding via the SLiMs helps them recognize and bind the regulatory proteins or the substrates to dephosphorylate them. PP1 is known to bind through RVxF, SILK and MyPhone motifs to its regulatory proteins (shown in gray). B56 regulatory subunit of PP2A binds through LxxIxE motifs to its substrates (shown in gray). (C) Phosphatase activity can be regulated by modulation of the SLiM motif. Phosphorylation within or near the SliM sequence can lead to decreased phosphatase binding either by direct blocking in the case of PP1 or indirectly by binding to phospho-binding 14-3-3 proteins to block the site of interaction. In case of PP2A-B56 SLiM, this phosphorylation can enhance phosphatase activity toward substrate by increasing the affinity for phosphorylated SLiM.