Elsevier

Cellular Signalling

Volume 20, Issue 11, November 2008, Pages 2059-2070
Cellular Signalling

Myosin phosphatase interacts with and dephosphorylates the retinoblastoma protein in THP-1 leukemic cells: Its inhibition is involved in the attenuation of daunorubicin-induced cell death by calyculin-A

https://doi.org/10.1016/j.cellsig.2008.07.018Get rights and content

Abstract

Reversible phosphorylation of the retinoblastoma protein (pRb) is an important regulatory mechanism in cell cycle progression. The role of protein phosphatases is less understood in this process, especially concerning the regulatory/targeting subunits involved. It is shown that pretreatment of THP-1 leukemic cells with calyculin-A (CL-A), a cell-permeable phosphatase inhibitor, attenuated daunorubicin (DNR)-induced cell death and resulted in increased pRb phosphorylation and protection against proteolytic degradation. Protein phosphatase-1 catalytic subunits (PP1c) dephosphorylated the phosphorylated C-terminal fragment of pRb (pRb-C) slightly, whereas when PP1c was complexed to myosin phosphatase target subunit-1 (MYPT1) in myosin phosphatase (MP) holoenzyme dephosphorylation was stimulated. The pRb-C phosphatase activity of MP was partially inhibited by anti-MYPT11–296 implicating MYPT1 in targeting PP1c to pRb. MYPT1 became phosphorylated on both inhibitory sites (Thr695 and Thr850) upon CL-A treatment of THP-1 cells resulting in the inhibition of MP activity. MYPT1 and pRb coprecipitated from cell lysates by immunoprecipitation with either anti-MYPT1 or anti-pRb antibodies implying that pRb–MYPT1 interaction occurred at cellular levels. Surface plasmon resonance-based experiments confirmed binding of pRb-C to both PP1c and MYPT1. In control and DNR-treated cells, MYPT1 and pRb were predominantly localized in the nucleus exhibiting partial colocalization as revealed by immunofluorescence using confocal microscopy. Upon CL-A treatment, nucleo-cytoplasmic shuttling of both MYPT1 and pRb, but not PP1c, was observed. The above data imply that MP, with the targeting role of MYPT1, may regulate the phosphorylation level of pRb, thereby it may be involved in the control of cell cycle progression and in the mediation of chemoresistance of leukemic cells.

Introduction

The retinoblastoma protein (pRb) is the product of the retinoblastoma susceptibility gene and it functions as a key regulator of cell cycle progression [1]. The pRb acts as a growth suppressor and exerts its effect via binding to a variety of cellular proteins. Interaction of pRb with the E2F family transcription factors is the best characterized and it is involved in the suppression of the synthesis of proteins necessary to progression of the cell cycle from G1 to S phase. Phosphorylation of pRb at several sites controls its binding to regulatory proteins [2]. In hypophosphorylated state, pRb interacts with and inactivates E2F transcription factors. Hyperphosphorylation of pRb by cyclin-dependent kinases relieves this inhibition by dissociating E2F from pRb. It has been shown that cyclin D/CDK4 and cyclin A/E/CDK2 complexes are involved in pRb phosphorylation [3]. The phosphorylation state of pRb influences its proteolytic degradation during cell death induced by chemotherapeutic drugs [4]. It has been shown that drug-induced apoptosis of cancer cells is accompanied with the activation of Ser/Thr-specific protein phosphatases followed by pRb dephosphorylation and proteolytic degradation via caspase-3 [4], [5].

The mechanism of the dephosphorylation of pRb is less understood, especially concerning the regulatory/targeting subunits of protein phosphatases involved [6]. Studies with cell-permeable inhibitors of protein phosphatase, such as calyculin-A (CL-A), okadaic acid (OA) and tautomycin (TM), have established that protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) may be involved in the control of the phosphorylation level of pRb [7]. Inhibition of PP2A affects pRb phosphorylation via decreasing the activity of cyclin-dependent kinases. Interaction of pRb with PP1 catalytic subunit (PP1c) isoforms [8], [9] and dephosphorylation of several sites in pRb by PP1c has been demonstrated [5], [10]. Consistent with these observations, PP1cα inhibition was required to permit G1/S transition during the cell cycle [11], whereas PP1cα activation prevented oncogenic transformation of the cells via regulation of the phosphorylation level of pRb [12]. PP1 is present as holoenzyme forms in the cells in which PP1c is complexed to inhibitory or regulatory subunits. The function of many PP1c-binding regulatory proteins is to target PP1c to the substrate and/or to different subcellular compartments of the cell [13]. High molecular mass PP1 forms were shown to dephosphorylate pRb in gel-filtered cell lysate, however the putative regulatory/targeting proteins have remained to be identified [14]. pRb is largely confined to the nucleus, therefore the nuclear PP1c-binding proteins such as nuclear inhibitor of PP1 (NIPP1) of 41 kDa [15] and the phosphatase nuclear targeting subunit (PNUTS) of 110 kDa [16] were assumed as possible regulatory proteins. Consistent with this idea, PNUTS was found to inhibit pRb dephosphorylation by PP1c [17]. Myosin phosphatase (MP), a PP1 type holoenzyme, is well characterized in respects of its role in the dephosphorylation of myosin and in the regulation of cell contractility [18]. MP has been localized in the cytoplasm and also in the nucleus of different cells [19], [20], [21]. The latter indicates that in addition to being a mediator of contractile processes, MP might function in other signaling pathways as well.

MP holoenzyme consists of PP1c, a large regulatory subunit (110–130 kDa) termed myosin phosphatase target subunit-1 (MYPT1), and a 20 kDa subunit of yet unknown function [22], [23]. MP is involved in the regulation of smooth muscle contraction via controlling the phosphorylation level of the 20 kDa light chain of myosin (MLC20). MYPT1 targets PP1c to myosin and stimulates the dephosphorylation of MLC20. Phosphorylation of MYPT1 with Rho-kinase on Thr695 [24] and/or Thr850 [25] results in the inhibition of the phosphatase activity of MP. Another mechanism for the inhibition of MP is its interaction with C-kinase activated phosphatase inhibitor of 17 kDa (CPI-17) which becomes inhibitory after phosphorylation by protein kinase C [26]. Transfection of MYPT1 as a GFP fusion protein (MYPT1-GFP) into different cell lines resulted in a predominant nuclear localization of the fusion protein and it abolished dramatically the viability of the cells [27]. Stable transfection was achieved only in HEK293 cells, where the normal pRb function was abrogated. These results raised the possibility that myosin phosphatase (MP) may be involved in the dephosphorylation of pRb, and that MYPT1 may target PP1c to pRb substrate. However, the interaction and spatial relationship of the native pRb and MYPT1 as well as the demonstration that phosphorylated pRb is a substrate of MP has remained to be elucidated.

Our present work shows that treatment of THP-1 leukemic cells with CL-A induces increased pRb phosphorylation and enhanced phosphorylation of MYPT1 at the inhibitory sites suggesting a parallel decrease in MP activity. In THP-1 cell lysates, MP accounts for more than 50% of the total pRb phosphatase activity. We prove that MYPT1 interacts with pRb and plays targeting role in directing PP1c toward the substrate during pRb dephosphorylation. Association of MYPT1 and pRb are identified by coprecipitation assays and by interaction analysis with surface plasmon resonance-based binding experiments. In addition, MYPT1 and pRb are shown to colocalize in the nucleus as revealed by confocal microscopy. In summary, our study identifies MYPT1 as a targeting subunit involved in pRb dephosphorylation and suggests that MP may be a major regulatory element in the control of the phosphorylation level of pRb. The above data imply that besides the well-defined role of MP in mediating cell contractility this enzyme may also be involved in the regulation of cell cycle progression.

Section snippets

Materials

Chemicals and vendors were as follows: [γ-32P]ATP (Hungarian Isotope Institute, Budapest, Hungary), Protein A Sepharose, nitrocellulose membrane (0.45 μm pore size); glutathione-sepharose (GE Healthcare Bio-Sciences AB, Uppsala, Sweden); amylose resin (New England Biolabs, Beverly, MA); Complete Mini Protease Inhibitor Cocktail Tablets (Roche Diagnostics, Mannheim, Germany); Antifade Light Kit, DAPI (Molecular Probes, Eugene, OR); penicillin/streptomycin solution, antibiotic–antimycotic

Survival of THP-1 cells treated with DNR in the absence or presence of CL-A

It was shown that drug-induced apoptosis of cancer cells was accompanied by the dephosphorylation-dependent degradation of pRb [4], [5]. To clarify the role of protein phosphatases in this process the influence of CL-A, a cell-permeable inhibitor of PP1 and PP2A, was examined on the survival of THP-1 cells treated with DNR. Fig. 1 shows that CL-A alone, applied at 10–100 nM concentration for 1 h, induced a moderate, but significant decrease in cell survival. The time interval for CL-A

Discussion

Our present data suggest that CL-A, a membrane-permeable inhibitory toxin of protein phosphatases antagonizes DNR-induced cell death of THP-1 leukemic cells in a concentration-dependent manner. CL-A alone (10 to 100 nM) induces cell death and its cytotoxic effect is more apparent at higher concentrations. Thus, in rescuing the cells from DNR-induced apoptosis, CL-A concentration is compromised at an intermediate dose (50 nM) at which its cytotoxic effect is relatively low. Our findings are

Conclusions

In conclusion, our present data suggest that MP is involved in the dephosphorylation of pRb, and its MYPT1 subunit targets PP1c toward phosphorylated pRb substrate. Since pRb phosphorylation plays crucial roles in the transition between the distinct phases of cell cycle, the interconverting enzymes may be the targets of various drugs developed for the modulation of cell cycle progression. In these respects, the cyclin-dependent kinases, that phosphorylate pRb, have been the center of interest

Acknowledgments

We thank Dr. Michael P. Walsh (Smooth Muscle Research Group, University of Calgary) for providing recombinant CPI-17. Thanks are due to Mrs. Ágnes Németh for excellent technical assistance. We are grateful to Dr. László Csernoch (Department of Physiology, University of Debrecen) for providing access and assistance to the confocal microscope. This work was supported by the following grants: Hungarian Scientific Research Fund (OTKA) K68416, K60620 and K49292 grants; Mecenatura Grant (07/2005) of

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