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 alpha 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-1/PP-2A inhibitor, calyculin A. Immunofluorescence 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.

Figure 1

Phosphorylation of PP-1C in vitro and detection by phosphorylation-state-specific antibodies. (A) Sf9 PP-1Cα (Left) and PP-1Cγ (Right) were phosphorylated by purified cdc2/cyclin B (New England Biolabs) and nonradioactive ATP for 90 min. The samples were resolved by SDS/PAGE (10% acrylamide) and immunoblotted with G-97 antibody (anti-T320 phosphorylation-state-specific PP-1Cα antibody). (B) Sf9 PP-1Cα was incubated with cdc2/cyclin B for various times in the presence of [γ-³²P]ATP (Left) or nonradioactive ATP (Right). Samples were resolved by SDS/PAGE. [³²P]-Labeled PP-1Cα was detected by autoradiography. Samples incubated with nonradioactive ATP were transferred to Immobilon-P, followed by immunoblotting with G-98 antibody and autoradiography. The region of the gel or immunoblot containing PP-1C is shown. No immunoreactivity was observed in any other part of the immunoblot.

Figure 2

Phosphorylation of PP-1C during the cell cycle. T cell hybridoma KMls-8.3.5 were synchronized at the G₁/S or G₂/M boundaries, and arrested cells were released by incubating with fresh medium for the indicated time periods. Equal amounts of protein (20 μg) were resolved by SDS/PAGE (10% acrylamide) and transferred to Immobilon-P. (A) Lysates from asynchronous (lane C), G₁/S, G₂/M phase cells, or cells released from drug treatment were analyzed by immunoblotting using G-97 antibody (PP-1C, M_r ≈ 37). (B) Total PP-1Cα was measured in the same samples using an anti-PP-1Cα antibody. The stages of the cell cycle were confirmed by fluorescence-activated cell sorter analysis (data not shown). PP-1Cα is present in ≈5-fold higher amounts than PP-1Cα in KMls cells (data not shown). The signal obtained with the G-97 antibody in KMls cells therefore represents predominantly PP-1Cα.

Figure 3

T320 phosphorylation in cells stably transfected with wild-type PP-1Cα or a PP-1CαT320A mutant. T cell hybridoma KMls were infected with retrovirus stocks expressing either ID4-tagged wild-type PP-1Cα (T320) or ID4-tagged PP-1Cα in which T320 was mutated to alanine (T320A). Single clones that expressed similar levels of ID4-tagged PP-1Cα or PP-1Cα T320A were analyzed in this experiment. (Left) Control asynchronous cells. (Right) Untransfected (−), wild-type PP-1Cα-transfected (T320), and PP-1Cα T320A-transfected (T320A) cells were synchronized at the G₂/M phase using 400 ng/ml nocodazole treatment for 16 h. Whole cell lysates were prepared as described and aliquots (20 μg protein) were resolved on SDS/PAGE, followed by immunoblotting with either G-97 antibody (A) or anti-PP-1Cα antibody (B). The ID4-tagged PP1C (T-PP-1Cα) migrated with a slightly higher molecular weight than endogenous PP1Cα.

Figure 4

Effect of phosphatase and cdk kinase inhibitors on the T320 phosphorylation of PP-1C. Asynchronous cells (lane 1) or NIH 3T3 cells synchronized at the G₂/M phase using 400 ng/ml nocodazole treatment for 16 h (lanes 2–6) were incubated for an additional 1 h in the absence of inhibitors (lanes 1 and 2), in the presence of 300 μM olomoucine (lane 3), 100 nM okadaic acid (lane 4), 100 nM calyculin A (lane 5), or 300 μM olomoucine plus 100 nM calyculin A (lane 6). Whole cell lysates were prepared as described, and aliquots (15 μg protein) were resolved on SDS/PAGE and immunoblotted with either G-97 antibody (A) or anti-PP-1Cα antibody (B). Quantitation of three individual experiments indicated the following averages: control, 2 (arbitrary) units; nocodazole, 45 units; olomoucine, 4 units; okadaic acid, 46 units; calyculin A, 141 units; calyculin A plus olomoucine, 140 units.

Figure 5

T320 phosphorylation of PP-1Cα analyzed in NIH 3T3 cells by indirect immunofluorescence. NIH 3T3 cells were fixed, permeabilized, and stained with G-97 antibody followed by anti-rabbit IgG conjugated with Texas red as secondary antibody. (A) Low magnification view showing several cells at various stages of the cell cycle. (B–F) Higher magnification showing individual cells in prophase (B), prometaphase (C), metaphase (D), anaphase (E), and late anaphase (F). (A′–F′) DAPI-stained DNA of cells shown in A–F. The yellow arrowheads in A′ indicate 2 of 13 cells that are mitotic. The differential staining of mitotic cells is not due to cell thickness because identical results were obtained using confocal microscopy (data not shown). The results shown are representative of many individual experiments in which the same results were obtained.

Figure 6

Subcellular localization of PP-1Cα. KMls cells were synchronized at the G₂/M phase, pelleted, and fractionated; W, S, and P indicate whole cell lysates, soluble, and particulate fractions, respectively. Aliquots (20 μg protein) were subjected to SDS/PAGE and immunoblotting with either G-97 antibody (Left) or anti-PP-1Cα antibody (Right). Identical results were obtained for the subcellular distribution of PP-1Cγ.