PP2A Inactivation Research Articles

A Dynamic Equilibrium between CDKs and PP2A Modulates Phosphorylation of pRB, p107 and p130

It is thought that G1 cyclin/CDK mediated phosphorylation of pocket proteins from mid G1 to mitosis is reversed via dephosphorylation in mitosis. We examined the mechanisms involved in the unexpectedly rapid dephosphorylation of the pocket proteins induced via inhibition of cellular protein synthesis by cycloheximide (CHX) as well as direct inhibition of CDKs by flavopiridol. CHX and flavopiridol-induced dephosphorylation of pocket proteins is attributable to inactivation of D-type cyclin/CDKs and G1/S CDKs, respectively, which unmasks a phosphatase activity that targets the three pocket proteins apparently throughout the cell cycle. Treatment of cells with phosphatase inhibitors at concentrations selective for PP2A inhibition prevents CHX and flavopiridol-mediated dephosphorylation of pocket proteins in vivo. Also, ectopic expression of SV40 small t antigen, which inhibits PP2A via disruption of trimeric PP2A holoenzymes, delays CHX-induced pocket protein dephosphorylation. Moreover, dephosphorylation of p130 and p107 in cell extracts is inhibited by concentrations of okadaic acid known to inhibit PP2A, but not PP1. Finally, the PP2A catalytic subunit (PP2A/C) specifically interacts with both p130 and p107 in quiescent cells as well as cells progressing throughout the cell cycle. Together, these results demonstrate that the overall phosphorylation state of pocket proteins is determined, at least in part, by a dynamic equilibrium between CDKs and PP2A, or a closely related PP2A-like enzyme. These findings have important implications, as cell cycle or checkpoint-dependent inhibition of CDK activities counteracted by an active PP2A should have imminent effects on the phosphorylation state and activities of pocket proteins. PLEASE SEE ERRATUM REGARDING FIGURE 3D AT THIS LINK: http://www.landesbioscience.com/journals/cc/abstract.php?id=1596

Interleukin-6 increases rat metalloproteinase-13 gene expression through Janus kinase-2-mediated inhibition of serine/threonine phosphatase-2A

Interleukin-6 (IL-6) increases metalloproteinase-13 (MMP-13) gene expression by increasing phosphorylated c-Jun and by inhibiting serine/threonine phosphatase-2A (PP2A) activity. We investigated the mechanisms by which IL-6 induces c-Jun phosphorylation and PP2A inactivation in Rat-1 fibroblasts. We show that IL-6 increased MMP-13 mRNA, phosphorylated c-Jun, and activator protein 1 (AP1) binding activity without increasing c-Jun-N-terminal kinase (JNK) activity. These effects did not seem to be mediated by ERK, p38 MAP kinase, phosphatidylinositol-3-kinase, calmoduline-dependent protein kinase, protein kinase C (PKC) or protein kinase A since inhibition with specific inhibitors did not abrogate these effects. IL-6 increases PP2A catalytic subunit tyrosine phosphorylation. Inhibition of the tyrosine kinase Jak2, with the specific inhibitor AG490, abrogated this effect. Likewise, this Jak2 inhibitor blocked the effects of IL-6 on c-Jun phosphorylation, AP1 binding activity and metalloproteinase-13 gene expression. We conclude that IL-6 increases MMP-13 gene expression by activation of Jak2, resulting in tyrosine phosphorylation of the catalytic subunit of PP2A, which in turn decreases PP2A activity and prolongs c-Jun phosphorylation.

Protein phosphatases independently regulate vesicle movement and microtubule subpopulations in hepatocytes.

To investigate the regulation of microtubule (MT)-based vesicle transport and the interphase MT array in hepatocytes, we have used okadaic acid (OKA) and microcystin (MCYST), two toxins that inhibit serine-threonine protein phosphatases (PP) 1 and 2A, to alter cellular phosphorylation. Video-enhanced differential interference contrast microscopy analysis revealed that both toxins inhibited the frequency, velocity, and run length of MT-dependent vesicle movements dose dependently between 50 and 500 nM. At our maximum dose of 500 nM, both toxins significantly decreased PP2A activity (OKA to 45 +/- 12% and MCYST to 57 +/- 2%), whereas PP1 was inhibited only by MCYST. Because no additional effects on vesicle movements were caused by MCYST over the changes caused by OKA, these data implicate PP2A in the regulation of MT-dependent vesicle movement. To understand whether the changes in parameters of vesicle movements were due to changes in the MT array, the effects of these toxins on MT distribution were examined by immunofluorescence microscopy. Although lower doses of OKA produced no effects, treatment with 500 nM OKA altered MT organization and also caused fragmentation and loss of acetylated (stable) MTs. In contrast, MCYST concentrations up to 500 nM elicited no changes in MT organization in general or in the acetylated (stable) array. From these findings we conclude that inhibition of MT-dependent vesicle movement by the PP inhibitors, MCYST and OKA, in hepatocytes cannot result from changes or disruption in the MT array. Because OKA (an inhibitor of PP2A only in our system) at high doses caused loss of stable MTs, whereas MCYST (an inhibitor of both PP1 and PP2A) did not, we conclude that the control of the preservation of the stable MT array in hepatocytes is complex. Stable MTs require active PP2A for maintenance, but the disruption of the array through inhibition of PP2A can be prevented if PP1 is also inhibited, suggesting that the relative degree of phosphorylation of multiple cellular components is the determinant of MT stability.

Role of Protein Phosphatase 2A in the Regulation of Mitogen-Activated Protein Kinase Activity in Ventricular Cardiomyocytes

Incubation of cultured, neonatal rat ventricular cardiomyocytes with 100 nM phorbol 12-myristate-13-acetate (PMA) induced a transient suppression of PP2A activity at 5 min, an effect that was reversed after 15 min of exposure to PMA. This inactivation was correlated with a transient increase in the phosphorylation level of the catalytic subunit of PP2A (193 ± 38% of control levels at 5 min). Simultaneously to the transient inactivation of PP2A, we observed a rapid and reversible phosphorylation of 42-kDa MAP kinase (474 ± 65% of control levels at 5 min, and 316 ± 44% at 15 min) in cardiomyocytes treated with PMA. This transient phosphorylation was accompanied by a transient increase in cytosolic MAP kinase activity (209 ± 17% of control values at 5 min and 125 ± 7% at 15 min). Okadaic acid (1 μM) completely blocked the decrease in the phosphorylation level and activity of MAP kinase occurring after 5 min of exposure to PMA. These data demonstrate that PP2A inactivation and MAP kinase activation are very strongly correlated in cardiomyocytes, indicating that PP2A plays a negative modulatory role in the regulation of MAP kinase activity.