Phosphorylation Dependent Sequestration Of Protein Phosphatase-1 I (PP-1 I ) By Protein 14-3-3

. PP-1 is involved in the control of the meiotic and mitotic cell divisions at the G2 to M transition by dephosphorylating phospho-serine 217 (phospho-serine 287 in Xenopus) of Cdc25, accompanied by activation of Cdc25 which would then dephosphorylate and activate Cdk1 . PP-1 I has been proposed to be the form of PP-1 that dephosphorylates and activates Cdc25. Here we show that PP-1 I is sequestered in a phosphorylation dependent manner by protein 14-3-3 involving phosphorylation of serine 71 of I-2 moiety of PP-1 I by C-TAK1. Phosphorylation of serine 71 of the I-2 moiety of PP-1 I by C-TAK1 enhanced the sequestration and inhibition of PP-1 I by Protein 14-3-3. Phosphorylation of serine 71 of the I-2 moiety of PP-1 I caused an increase in the Stability Energy, Binding Energy of the interaction between PP-1 I and Protein 14-3-3 and a significant decrease in the IC 50 of the inhibition of PP-1 I by Protein 14-3-3. PP-1 I regulation by C-TAK1 and Protein 14-3-3 is a mechanism for the control of Cdc25 at the G2 to M transition of the cell cycle.


Abstract.
PP-1 is involved in the control of the meiotic and mitotic cell divisions at the G2 to M transition by dephosphorylating phospho-serine 217 (phospho-serine 287 in Xenopus) of Cdc25, accompanied by activation of Cdc25 which would then dephosphorylate and activate Cdk1 . PP-1I has been proposed to be the form of PP-1 that dephosphorylates and activates Cdc25. Here we show that PP-1I is sequestered in a phosphorylation dependent manner by protein 14-3-3 involving phosphorylation of serine 71 of I-2 moiety of PP-1I by C-TAK1. Phosphorylation of serine 71 of the I-2 moiety of PP-1I by C-TAK1 enhanced the sequestration and inhibition of PP-1I by Protein 14-3-3. Phosphorylation of serine 71 of the I-2 moiety of PP-1I caused an increase in the Stability Energy, Binding Energy of the interaction between PP-1I and Protein 14-3-3 and a significant decrease in the IC50 of the inhibition of PP-1I by Protein 14-3-3. PP-1I regulation by C-TAK1 and Protein 14-3-3 is a mechanism for the control of Cdc25 at the G2 to M transition of the cell cycle.

Introduction.
Protein phosphatase-1 (PP-1) was originally identified as an enzyme that dephoshorylates enzymes involved in the control of Glycogen breakdown and synthesis, including Proteins" because they apparently target the catalytic subunit of PP-1, PP-1C to different cellular compartments where PP-1C can dephosphorylate its many substrates [43,44]. It has also been argued that PP-1 is regulated by "Protein Targeting Mechanism" (PTM) as opposed to the established regulation of enzymes by activating proteins and inhibitory proteins, "Activating and Inhibitory Mechanism" (AIM) [43.44]. There is no scientific evidence that supports the regulation of PP-1 by the PTM as the PTM implies that the concentration of PP-1C would be a lot higher than what it is currently estimated to be [45]. Considering that there may be more than 100 different substrates for PP-1C, under PTM, the concentration of PP-1C in the cell would have to be at least one hundred times more than what it is currently estimated to be. It must be noted that there is not a single study that has determined the concentration of PP-1C in the cell with any degree of accuracy. On the other hand, the regulation of PP-1 by AIM follows established mechanism of enzyme regulation: PP-1 is regulated by Activating and Inhibitory Proteins and its concentration in the cell does not need to be proportionally higher in order to account for the dephosphorylation of all of its substrates in the different cellular compartments where its substrates are found [45].
A major proportion of PP-1 in the cell is in the form of PP-1I which is a complex consisting of PP-1C and I-2, and other regulatory proteins [46][47][48][49][50][51][52]. PP-1I is the same enzyme as ATP-Mg dependent protein phosphatase [53][54][55][56][57]. For a number of years, it was thought that PP-1I and ATP-Mg dependent protein phosphatase are two distinct enzymes because the ATP-Mg dependent protein phosphatase was described as a Protein phosphatase consisting of a single protein of apparent molecular mass 70 kDa [55,56]  directed and not enzyme directed because 32 P-BAD was used as substrate and Protein 14-3-3 is known to bind to BAD phosphorylated by PKA [61]. How Protein 14-3-3 regulates the activity of PP-1I remains to be established. Examination of the motif surrounding phosphorylation site threonine 72 of I-2 (DEPSpTP) reveals that it is a phosphorylation dependent binding and sequestration site for Protein 14-3-3 [62-67]. Serine 71 of I-2 can be phosphorylated by C-TAK-1 and the motif DEPpSTP in I-2 is very similar to a consensus C-TAK1 phosphorylation motif (RXXpSXP) [62][63][64][65][66][67]. The motif in I-2 (DEPpSTP) is very similar to DXXpSLP which has been shown to moderately bind Protein 14-3-3 [67]. It is not clear how Protein 14-3-3 sequesters PP-1I to inhibit PP-1I.

Materials and Methods.
Phosphorylase b, Phosphorylase kinase and PKA were purified from pig skeletal muscle as described in [82,83]. PP-1I and PP-1I-ACK were purified brain as described in The full structure of PP-1I has not been determined from X-ray crystallography because I-2 has very little ordered structure. Only 59 residues of I-2 was observed from the crystal lattice of PP-1I reconstituted from recombinant PP-1C and I-2. [87]. The structure of PP-1I has been rendered from partial crystallographic data, NMR data and SAXS data.
However, these studies have not been verified and did not take into account phosphorylation of serine 71 of I-2 [88]. In the present work, the structure of I-2 was rendered de novo computationally pursuant to Xu and Zhang [89,90]. The structure of Human PP-1C was also rendered de novo computationally pursuant to Yang et al. [91,92].
The structure of Protein 14-3-3 as determined by Smidova et al. [93] was used in this study.
For the purpose of thermodynamic calculations, the phosphorylation of serine 71 of I-2 moiety in the PP-1I complex was performed using the Build Model Program of FoldX pursuant to Guerois et al. and Schymkowitz et al. [94,95]. The sequestration and binding of PP-1I by Protein 14-3-3 was performed by Docking Experiments using Z Dock Program pursuant to Pierce et al. [96]. The structures of PP-1C, I-2, PP-1I, and PP-1I-Protein 14-3-3 were analyzed and visualized by the CCP4 Molecular Graphics Program Version 2.10.11 as described by Mc Nicolas et al [97] and the ZMM Molecular Modeling Program as described by Garden and Zhorov [98]. Determination and calculation of Stability Energy (ΔGstability energy) of the protein complexes and non-complexed proteins was performed using the Stability Program of FoldX as described by Guerois et al. and Schymkowitz et al. [94,95]. Binding Energy (ΔΔGbinding energy) of protein complex was determined and calculated using the Analyze Complex Program of FoldX as described by Guerois et al. and Schymkowitz et al. [94,95]. Binding Energy Difference (ΔΔΔGbinding energy difference) between phospho-PP-1I-Protein 14-3-3 complex and dephospho-PP-1I-Protein 14-3-3 complex was calculated pursuant to Teng et al. and Nishi et al. [99][100][101] from the equation: ΔΔΔGbinding energy difference = ΔΔGbinding energy (binding energy of phospho-PP-1I-14-3-3 complex) -ΔΔGbinding energy (binding energy of phospho-serine 71-PP-1I -14-3-3 complex.

Results.
The complete structure of PP-1I has not been determined from X-ray crystallography because I-2 apparently has very little ordered structure and only 59 residues of rabbit I-2 could be observed from the crystal lattice of PP-1I reconstituted from recombinant rabbit PP-1C and I-2 [87]. A PP-1I structure was rendered from partial crystallographic data, NMR data and SAXS data. However, these studies have not been verified and phosphorylation of serine 71 was not taken into account in these studies [88]. The structure of Human I-2 which has 90% identity with Rabbit I-2 was rendered de novo  (Table 1) indicating that phospho-serine 71 does not affect the activity of PP-1I.
It was previously shown that PP-1I purified from brain is found in a complex consisting of PP-1C and I-2 and other proteins including Protein 14-3-3 which inhibits the activity of PP-1I (most probably substrate directed and not enzyme directed) [52]. C-TAK1 can phosphorylate serine 71 of I-2 within the phosphorylation recognition motif DEPpSTP.  (Table 1). These results are consistent with the conclusion that phosphorylation of serine 71 of I-2 enhances the stability and binding affinity between PP-1I and Protein 14-3-3. Figure 5A shows that phosphorylation of serine 71 of I-2 moiety in PP-1I enhances its inhibition by Protein 14-3-3 by causing a decrease of I50 from >300 µM to ~0.03 µM.
Phosphorylation of serine 71 of I-2 moiety in PP-1I did not have a significant effect on the activation of PP-1I by PP-1I-ACK ( Figure 6).

Discussion.
PP-1I is a major form of PP-1 PP-1I represents almost 30% of total PP-1 activity in extracts of brain from freshly killed pigs PP-1I consists minimally of PP-1C and I-2 and other regulatory proteins, including Protein 14-3-3 and C-TAK1. PP-1C has been proposed to be a key controller of meiosis and mitosis because (i) inhibiting its activity antagonizes G2 to M transition of the cell cycle [7-11], (ii) it is regulated by Cdk-1 [58,59], and (iii) it is regulated by Protein 14-3-3 and C-TAK1 [13-15 and present work].
In the present work, structure model analysis and thermodynamic calculations show that PP-1I can be sequestered by Protein 14-3-3 by a phosphorylation dependent mechanism involving phosphorylation of serine 71 of its I-2 moiety. Phosphorylation of serine 71 of the I-2 moiety of PP-1I caused an increase in the Stability Energy and Binding Energy of the interaction between PP-1I and Protein 14-3-3. In vitro enzyme assay of PP-1I shows that phosphorylation of reconstituted PP-1I by C-TAK1 significantly enhanced its inhibition by Protein 14-3-3. It was previously shown that PP-1I purified from brains of freshly killed pigs was complexed to Protein 14-3-3 and C-TAK1 and using 32 P-BAD phosphorylated by PKA as substrate, reconstituted PP-1I was inhibited by Protein 14-3-3 in vitro [52]. The effect of Protein 14-3-3 on the dephosphorylation of 32 P-BAD by reconstituted PP-1I described in [52] is most probably substrate directed and not enzyme directed because Protein 14-3-3 is known to bind to phosphorylated BAD and PP-1I was inhibited with I50 of more than 10 µM when the concentration of 32 P-BAD in the assay was ~5 µM. In the present work, it is shown that phoshorylation of serine 71 by C-TAK1 enhances the inhibition of PP-1I by Protein 14-3-3 by decreasing the I50 from >300 µM to ~0.03 µM when the concentration of the substrate, 32 P-labeled Phosphorylase kinase was ~5 µM. The results of the present work are consistent with the conclusion that the effects of Protein 14-3-3 on the dephosphorylation of 32 P-labeled Phosphorylase kinase was enzyme directed and not substrate directed.
Phosphorylation of serine 71 of the I-2 moiety of reconstituted PP-1I by C-TAK1 did not cause any significant inhibition of reconstituted PP-1I in stark contrast to a report by Platholi et al. [102]. There are major problems in the data presented in the paper by Tung. The first author of the paper by Platholi et al. [102] was a student working under the supervision of Dr H.Y. Lim Tung. In the Acknowledgements Section of their paper, Platholi et al. [102] have admitted that they stole the data showing that C-TAK1 phosphorylated serine 71 of I-2 from Dr H.Y. Lim Tung who was never informed that his scientific data was being used without his knowledge and permission. The authors in [102] are lying when they reported that they purified PP-1I from brains of freshly killed pigs and prepared 32 P-labeled Phosphorylase a. PP-1I and 32 P-labeled Phosphorylase a described in [102] were purified and prepared respectively by Dr H.Y. Lim Tung. The authors of the paper by Platholi et al. [102] did not and does not know how to purify PP-1I from brains of freshly killed pigs. The other data in the paper by Platholi et al. [102] showing the so-called effect of PFTAIRE kinase on reconstituted PP-1I are all fabricated and falsified. PFTAIRE protein kinase is Cyclin dependent protein kinase requiring Cyclin D for full activity [103]. See [104] for a detailed discussion of why the paper by Platholi et al [102] is riddled with data fabrication and falsification.

Serine 71 of I-2 lies within a phosphorylation recognition motif (DEPpSTP) for C-TAK1.
C-TAK1 is an enzyme which was first identified for its ability to phosphorylate serine 217 of Cdc25 which forms a binding and sequestration site for Protein 14-3-3 [62,63].
The sequestration of Cdc25 by Protein 14-3-3 has been proposed to be an obligatory control that prevents G2 to M transition of the cell cycle [12][13][14]. De-sequestration of Cdc25 by Protein 14-3-3 involves a very complex mechanism that remains to be fully elucidated. It has been proposed that a form of PP-1 is involved in the dephosphorylation of serine of Cdc25 [12][13][14]. The finding that the I-2 moiety of PP-1I was phosphorylated by C-TAK1 and that phosphorylation of serine 71 of the I-2 moiety of PP-1I by C-TAK1 enhanced its sequestration and inhibition by Protein 14-3-3 provides a linkage between PP-1I and the control of G2 to M transition of the cell cycle. It is submitted that PP-1I is involved in the dephosphorylation of phospho-serine 217 serine (phospho-serine 287 in Xenopus) of Cdc25, accompanied by the activation of Cdc25 which would dephosphorylate and activate Cdk1 at the G2 to M transition in mitotic cell division [15].
With respect to meiotic cell division, the activity PP-1I could be controlled by its specific inhibitors, including I-1 and heat-stable protein inhibitors first identified by Boyer et al. [15,105].     (1 x 60 cm). The eluates containing phosphorylated PP-1I were concentrated by vacuum dialysis. Unphoshorylated PP-1I was treated in a similar fashion except that C-TAK1 was replaced with bovine serum albumin. The stoichimetry of phosphorylation was determined by using 32 P-labeled ATP instead of ATP:it was determined that ~0.83 mole 32 P-phosphate was incorporated per mole of PP-1I. The activities of dephosho PP-1I and phospho PP-1I were assayed by determining the release of 32 P-phosphate from 32 P-Phosphorylase kinase at 10 µM in the presence of saturating amount of PP-1I-ACK, 0.125 mM ATP and 1.25 mM Mg 2+ . 100% PP-1I activity was equivalent to 0.01 unit (the amount of PP-1I that catalyzed the release of 0.01 nmol Pi/min when the concentration of the substrate was 10 µM).

Figure 6. Panel A, Specific Activity of Dephospho-serine 71-I-2-PP-1I (circles) and
Phospho-serine 71-I-2-PP-1I (squares). 40 µg reconstituted PP-1I was phosphorylated by incubation with 4 µg C-TAK1 in the presence of 5 mM Mg 2+ and 1.25 mM ATP for 30 minutes at 30 O C and then gel-filtered through a Superdex column (1 x 60 cm). The eluates containing phosphorylated PP-1I were concentrated by vacuum dialysis. Unphophophorylated PP-1I was treated in a similar fashion except that C-TAK1 was replaced with bovine serum albumin. The stoichimetry of phosphorylation was determined by using 32 P-labeled ATP instead of ATP: it was determined that 0. 93