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Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases
BBRC Biochemical and Biophysical Research Communications 306 (2003) 382–387 www.elsevier.com/locate/ybbrc
Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinasesq Ferenc Erd} odi,a,* Enik} o Kiss,a Michael P. Walsh,b Bjarki Stefansson,c Jing Ti Deng,b Masumi Eto,c David L. Brautigan,c and David J. Hartshorned a
Department of Medical Chemistry, Medical and Health Science Center, University of Debrecen, H-4012 Debrecen, Bem t er 18/B, Hungary b Smooth Muscle Research Group and Canadian Institutes of Health Research Group in Regulation of Vascular Contractility, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alta., Canada T2N 4N1 c Center for Cell Signaling, University of Virginia School of Medicine, Box 800577-MSB 7225, Charlottesville, VA 22908, USA d Muscle Biology Group, University of Arizona, Shantz Bldg. 601, Tucson, AZ 85721, USA Received 9 May 2003
Abstract Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC50 values of 1–8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC50 ¼ 0:1 nM) and MPH (IC50 ¼ 8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: C-kinase-enhanced (potentiated) phosphatase inhibitors (CPI-17 and KEPI); Myosin phosphatase; Protein kinase A; Protein kinase G
Protein phosphorylation is a widespread regulatory mechanism and phosphorylation levels are a reflection of the balance between kinase and phosphatase activities, which can themselves be subjected to regulation. Cellular protein phosphatase-1 (PP1) activity is controlled by interaction with various targeting subunits
q
Abbreviations: CPI-17, C-kinase potentiated 17 kDa inhibitor of type-1 protein phosphatase; ILK, integrin-linked kinase; KEPI, kinase-enhanced protein phosphatase type-1 inhibitor; LC20, 20 kDa light chain of smooth muscle myosin II; MPH, myosin phosphatase holoenzyme; MYPT1, myosin phosphatase target subunit; PHI, phosphatase holoenzyme inhibitor; PKA, cAMP-dependent protein kinase (protein kinase A); PKG, cGMP-dependent protein kinase (protein kinase G); PP1, type-1 protein phosphatase; PP1c, catalytic subunit of PP1; ROK, Rho-associated kinase; I-1, inhibitor-1; I-2, inhibitor-2; DARPP-32; dopamine and cAMP regulated 32 kDa phosphoprotein. * Corresponding author. Fax: +36-52-412566. E-mail address: [email protected] (F. Erd} odi).
and PP1 inhibitory proteins (reviews [1–4]). PP1 inhibitory proteins have been known for decades. The earlier discoveries included inhibitors of type-1 protein phosphatase (PP1): inhibitor 1 (I-1), inhibitor-2 (I-2), and DARPP-32. I-1 and DARPP-32 are phosphorylated by cAMP-dependent kinase (PKA), which markedly increases the inhibitory potency. More recently, a PP1 inhibitor, called CPI-17 (a 17 kDa PKC-potentiated inhibitory protein of PP1), was discovered in porcine aorta [5]. CPI-17 is exclusively expressed in smooth muscles and in brain [6–8]. It was shown that phosphorylation of T38 by PKC increased inhibitory potency over 1000-fold [6]. Subsequently, orthologues of CPI-17, called PHI-1 (phosphatase holoenzyme inhibitor-1) [9] and KEPI (kinase-enhanced protein phosphatase-1 inhibitor) [10], were identified in mammalian tissues. PHI-1 is ubiquitously expressed among tissues, whereas KEPI is predominantly expressed in brain, heart, and muscle. Sequences around the inhibitory sites
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00976-8
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of phosphorylation in these proteins are conserved: 35 ARVTV40 K for CPI-17, 54 GKVTV59 K for PHI-1, and 69 GKVTV74 K for KEPI, i.e., consensus sequences for PKC. Indeed, PHI-1 and KEPI are also activated by phosphorylation with purified PKC [6,9,10]. Recently, in a study carried out with intact carotid artery it was found that prolonged exposure to an NO donor resulted in phosphorylation of CPI-17, suggesting involvement of PKG and/or PKA in regulation of the CPI-17 family [11]. One of the systems in which there is considerable interest in signaling pathways involved in regulation of phosphatase activity is smooth muscle. Phosphorylation of the myosin regulatory light chains (LC20) is an important regulatory mechanism and activation or inhibition of phosphatase leads to decreased or increased levels of myosin phosphorylation, respectively. In vascular muscle it is thought that agonist stimulation leads to inhibition of phosphatase and increased contraction at sub-optimal Ca2þ concentrations, whereas elevated cyclic nucleotides promote an opposite effect [12]. The myosin phosphatase holoenzyme (MPH) is composed of three subunits: a catalytic subunit PP1c (d isoform) and two non-catalytic subunits of approximately 18–20 kDa (M20) and 106–110 kDa [13]. The latter is termed myosin phosphatase target subunit, MYPT1. Two mechanisms have been proposed for inhibition of MPH: one via phosphorylation of MYPT1 by, e.g., ROK [14], and the other by phosphorylated CPI-17 [15]. Previously it was shown that I-1 and I-2 were not effective inhibitors of MPH [16] but that CPI-17 was effective (IC50 of 12 nM) [17]. PHI-1 can also inhibit MPH, although the potency of PHI-1 (IC50 of 30 nM) is lower than that of CPI-17 [9]. The effect of KEPI on MPH has not yet been determined. In addition to PKC, several kinases have been shown to phosphorylate CPI-17 and potentiate inhibition [18– 21]. ILK was shown to phosphorylate both CPI-17 and PHI-1 [21]. Phosphorylated CPI-17 and PHI-1 exhibited increased phosphatase inhibitory potency and induced Ca2þ -sensitization of contraction of Triton X-100 demembranated rat-tail arterial smooth muscle. The influence of phosphorylation by different kinases (other than PKC) has not been studied with KEPI. Our objectives were to investigate possible phosphorylation of MPH inhibitors (focusing on CPI-17 and KEPI) by PKA and PKG, as well as ILK. To document effects of phosphorylation on phosphatase activity, we used native PP1c and MPH purified from skeletal and smooth muscles, respectively.
Materials and methods Materials. Chemicals and vendors were as follows: [c-32 P]ATP, Perkin–Elmer Life Sciences (Boston, MA, USA); ATP, Sigma (St. Louis, MO, USA); adenosine 50 -[c-thio]triphosphate (ATPcS), Cal-
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biochem (San Diego, CA, USA); nitrocellulose membrane (0.45 lm pore size) and enhanced chemiluminescence (ECL) reagent kit, Amersham Pharmacia Biotech (Piscataway, NJ, USA); and nitrocellulose membrane (0.20 lm pore size), Bio-Rad Laboratories (Hercules, CA, USA). All other chemicals used were the highest grade commercially available. Proteins. ILK [22] and MPH [23] were purified from chicken and turkey gizzard, respectively. Purification procedures for the following proteins are given in [24]: PP1 catalytic subunit (PP1c) from rabbit skeletal muscle, recombinant hexahistidine-tagged PP1c (d isoform), LC20 of gizzard myosin, and 32 P-labeled LC20. Hexahistidine-tagged wild-type (WT) and mutant CPI-17 (CPI-17WT , CPI-17T38A , and CPI1735–147 ) [25] and hexahistidine-tagged PHI-1 [9] were expressed and purified as described previously. Polyclonal antibodies that recognize phosphorylated T38 in CPI-17 (anti-CPI-17pT38 ) [26] and T57 in PHI-1 (anti-PHI-1pT57 ) [21] were used as described previously. The cDNA fragment of human KEPI was amplified by PCR methods using an IMAGE clone No. 4877511 as a template, purchased from Incyte Genomics (CA, USA), and inserted into pET30 vector as described previously [6]. Mutation of T73 of KEPI to A was performed using the QuickChange site-directed mutagenesis kit (Stratagene, CA, USA). Hexahistidine-tagged wild-type and mutant KEPI (KEPIWT and KEPIT73A ) were prepared from bacterial lysates as described previously [6]. PKA inhibitor peptide and horseradish peroxidase-coupled goat anti-rabbit immunoglobulin were from Sigma (St. Louis, MO, USA), PKG was from Calbiochem (San Diego, CA, USA), PKA and ROK (ROKa/ROCKII) were from Upstate Biotechnology (Lake Placid, NY, USA). Phosphorylation of PP1 inhibitory proteins. Phosphorylation of wild-type or mutant CPI-17, PHI-1 or KEPI (10 lM of each) was carried out in an assay buffer containing 30 mM Tris–HCl (pH 7.5), 85 mM KCl, 10 mM EGTA, 10 mM dithiothreitol, 1% Tween 80, 1 lM microcystin-LR, 10 mM MgCl2 , and 0.2 mM [c-32 P]ATP (100– 500 cpm/pmol) in the presence of 2 lg/ml PKA or PKG, or 20% (v/v) of ILK in a total volume of 50 or 100 ll. The reaction was started by addition of [c-32 P]ATP and 5 or 10 ll aliquots were removed and spotted on P81 paper discs. The filter papers were treated as described [27] and radioactivity was quantified in a scintillation counter. The stoichiometry of phosphorylation was calculated from the amount of 32 P incorporated into the proteins. Phosphorylation was also verified by autoradiography after separation of the proteins by SDS–PAGE. The gels were dried and exposed to X-ray film. Phosphorylation was also carried out in the absence of microcystin-LR and with replacement of [c-32 P]ATP with 0.2 mM ATPcS to thiophosphorylate the proteins. The thiophosphorylated proteins were used to assay the influence of phosphorylation of CPI-17 or KEPI on the activity of PP1c or MPH. Thiophosphorylated CPI-17 and KEPI were also used to assess inhibitory phosphorylation of T38 in CPI-17 or T73 in KEPI. CPI-17 and KEPI (or their phosphorylation site mutants T38A or T73A) were subjected to SDS–PAGE, and the proteins were transferred to nitrocellulose membrane and detected with phosphospecific antibodies: anti-CPI-17pT38 or anti-PHI-1pT57 . Anti-PHI-1pT57 , but not anti-CPI-17pT38 , recognized phosphorylated T73 in KEPI. Phosphatase assays. Phosphatase activity of skeletal muscle PP1c (0.5 nM), recombinant His-PP1cd (0.5 nM) or MPH was determined with 5 lM 32 P-labeled LC20 substrate as described previously [24]. Effectors (non-phosphorylated and phosphorylated CPI-17 and KEPI proteins) were preincubated with the phosphatase for 5 min, and then the reaction was initiated by addition of the substrate.
Results and discussion Phosphorylation of CPI-17 and two mutants by PKA is shown in Fig. 1A. Wild-type CPI-17 was phosphory-
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Fig. 1. Phosphorylation of CPI-17 by PKA and PKG. (A) PKA: (s) CPI-17WT ; (M) CPI-17T38A ; () CPI-1735–147 ; (d) CPI-17WT plus 10 lM PKA inhibitor peptide. (B) PKG: (s) CPI-17WT ; (M) CPI-17T38A ; () CPI-1735–147 . Data are means SE (n ¼ 3–6). (C) Autoradiograms (AU) and Western blots (WB) with anti-CPI-17pT38 of CPI-17WT and CPI-17T38A phosphorylated by PKA or PKG.
lated to 0.5 mol P/mol protein and this phosphorylation was inhibited completely by the peptide inhibitor of PKA (Fig. 1A), confirming phosphorylation by PKA rather than a contaminating kinase. Mutation of the inhibitory site of phosphorylation in CPI-17T38A resulted in a reduced level of phosphorylation by PKA (0.2 mol P/mol) but, surprisingly, did not eliminate phosphorylation (Fig. 1A), indicating that CPI-17 has more than one site of phosphorylation by PKA. A truncation mutant of CPI-17 lacking the first 34 residues (CPI1735–147 ) was phosphorylated to <0.1 mol P/mol, suggesting that phosphorylation at T38 was reduced by elimination of residues N-terminal to the consensus sequence. Earlier it was shown that ROK phosphorylated
CPI-17 at T38 [18]. In addition to T38, S12 of CPI-17 is phosphorylated by PKC [5] and MYPT1 kinase [20] at substoichiometric levels, suggesting that phosphorylation at T38 promoted phosphorylation at S12. Positive cooperative phosphorylation was not observed with sequential phosphorylation of CPI-17, initially by ROK followed by PKA (data not shown), in contrast to the expected T38 and S12 phosphorylation in CPI-17 by MYPT1 kinase [20]. Phosphorylation of CPI-17 by PKG is shown in Fig. 1B. Compared to PKA the phosphorylation rate is faster and the stoichiometry of phosphorylation higher (0.75 mol P/mol). Another difference relative to PKA is that the two mutants, CPI-17T38A and CPI-1735–147 , showed very low levels of phosphorylation with PKG (0.04 and 0.03 mol P/mol, respectively). Autoradiography (Fig. 1C) confirmed that wild-type CPI-17 was phosphorylated by both PKA and PKG and that CPI17T38A was phosphorylated only by PKA. Use of an antibody specific for phosphorylated T38 confirmed that both PKA and PKG phosphorylated the inhibitory site of CPI-17 (Fig. 1C). CPI-17 phosphorylated by either PKA or PKG inhibited the activity of rabbit skeletal muscle PP1c with IC50 values of 4.0 and 1.4 nM, respectively (Fig. 2). The lower potency of inhibition for CPI-17 phosphorylated by PKA probably reflects the lower level of phosphorylation at T38 (0.5 mol P/mol) compared to PKG-phosphorylated CPI-17 (0.75 mol P/mol). Nonphosphorylated wild-type CPI-17 and CPI-17T38A phosphorylated by PKA did not inhibit native PP1c (Fig. 2). It is interesting that MPH was also inhibited by CPI-17 phosphorylated by PKG, but with lower potency (IC50 ¼ 8:2 nM) compared to isolated PP1c (Fig. 2). These results lead to an unexpected conclusion, namely that PKA and PKG can both phosphorylate CPI-17 at the inhibitory site and effect inhibition of isolated PP1c and MPH. In these respects
Fig. 2. Inhibition of phosphatase activity by non-phosphorylated and thiophosphorylated CPI-17. PP1c with: (}) non-phosphorylated CPI17WT ; () CPI-17WT thiophosphorylated by PKG; (M) CPI-17WT thiophosphorylated by PKA; and (O) CPI-17T38A thiophosphorylated by PKA. Myosin phosphatase holoenzyme with: (j) CPI-17WT thiophosphorylated by PKG. Data are means SE (n ¼ 2–4).
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PKA and PKG are similar to several other kinases [6,18–21]. The sequence similarity around the inhibitory phosphorylation sites of CPI-17, PHI-1, and KEPI prompted a comparative study of phosphorylation of these proteins by PKA and PKG. With PHI-1 as substrate, very low levels of phosphorylation (<0.1 mol P/mol) by PKA or PKG were obtained (data not shown). Results with wild-type KEPI and a phosphorylation site mutant, KEPIT73A , are shown in Fig. 3A. Phosphorylation of wild-type and mutant KEPI by PKA was also observed and, surprisingly, the phosphorylation site mutant, KEPIT73A , was a slightly more effective substrate. Thus, it is unlikely that T73 in KEPI is a major site of phosphorylation by PKA. This was also confirmed by Western blots with anti-PHI-1pT57 (Fig. 3B): wild-type KEPI phosphorylated by PKA gave a faint signal indicating a low level of phosphorylation at T73. An alternative site of phosphorylation by PKA in KEPI is S157, as suggested previously [10]; this site fits the PKA consensus sequence (RKLSP). PKG did not phosphorylate either the wild type or mutant KEPI (Fig. 3A). Thus, unlike T38 of CPI-17, cyclic nucleotide-dependent kinases fail to phosphorylate T57 of PHI-1 or T73 of KEPI, despite conserved amino acid sequences. On the other hand, ILK phosphorylated wild-type KEPI (KEPIWT ) but not KEPIT73A (Fig. 3A). Because the sequence of the phospho-peptide used as antigen to
Fig. 3. Phosphorylation of KEPI by ILK and PKA, but not PKG. (A) ILK: (s) KEPIWT ; (d) KEPIT73A . PKA: (M) KEPIWT ; (O) KEPIT73A . PKG: () KEPIWT ; (j) KEPIT73A . Data are means SE (n ¼ 3). (B) Coomassie blue-stained SDS gel, autoradiograms (AU), and Western blots (WB) with anti-PHI-1pT57 of KEPIWT and KEPIT73A phosphorylated by ILK or PKA.
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produce anti-PHI-1pT57 is identical to the corresponding sequence in KEPI, anti-PHI-1pT57 cross-reacted with wild-type KEPI phosphorylated by PKC (data not shown). The antibody also bound to KEPIWT but not the T73A mutant phosphorylated by ILK on Western blots (Fig. 3B). Phosphoamino acid analysis showed phosphorylation at both S and T by PKC, whereas ILK specifically phosphorylated T in KEPI (data not shown). The results indicate specific phosphorylation of KEPI at T73 by ILK. The effects of non-phosphorylated and thiophosphorylated wild-type and mutant KEPI were assayed using native PP1c (from skeletal muscle), recombinant PPlcd (from bacterial expression), and MPH. Previously it was found that KEPI phosphorylated by PKC inhibited recombinant PP1c (using phosphorylase a as substrate) with an IC50 of 2.7 nM [10]. In Fig. 4 it is shown that unphosphorylated KEPI had an IC50 of 1.8 lM. Wildtype KEPI phosphorylated by ILK inhibited each PP1c preparation with IC50 values of 3.3 nM for recombinant PP1cd and 0.1 nM for native PP1c. The point to emphasize is that recombinant PP1cd is less sensitive to inhibition by KEPI (similar results were obtained with I-1 [28]). Furthermore, it is noteworthy that phosphoKEPI potently inhibits MPH with an IC50 value of 8.6 nM. Thus, in addition to CPI-17 and PHI-1, KEPI is a third inhibitor that can suppress the myosin phosphatase holoenzyme, although the holoenzyme is 80fold less sensitive to inhibition than isolated PP1c. Wild-type KEPI phosphorylated by PKA gave an IC50 of 17 nM (with rabbit PP1c) whereas KEPIT73A phosphorylated by PKA was similar to unphosphorylated KEPI (IC50 0:6 lM), suggesting that the putative S157 site is not inhibitory and the inhibition observed with PKA-phosphorylated KEPI is due to fractional phosphorylation at T73.
Fig. 4. Inhibition of phosphatase activity by nonphosphorylated and thiophosphorylated KEPI. PP1c with: (}) non-phosphorylated KEPIWT ; () non-phosphorylated KEPIT73A ; (s) KEPIWT thiophosphorylated by ILK; (M) KEPIWT thiophosphorylated by PKA; and (O) KEPIT73A thiophosphorylated by PKA. Recombinant PP1cd with: () KEPIWT thiophosphorylated by ILK. Myosin phosphatase holoenzyme with: (d) KEPIWT thiophosphorylated by ILK. Data are means SE (n ¼ 2–3).
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Kitazawa et al. [26] showed in femoral artery preparations that CPI-17 was phosphorylated in response to several stimulants including histamine (intact strips) and GTPcS (a-toxin permeabilized strips). Inhibitors of ROK and PKC both reduced contraction and CPI-17 phosphorylation, suggesting the involvement of at least two signal transduction pathways. Thus, under these conditions the phosphorylation of CPI-17 favored an increased contractile response, as expected. Other results consistent with the proposed role of CPI-17 relative to myosin phosphatase activity are that addition of nitroprusside (an NO donor) to histamine-contracted carotid artery resulted in relaxation via decreased CPI-17 phosphorylation and increased myosin phosphatase activity [11]. However, an intriguing observation was that prolonged exposure to nitroprusside resulted in phosphorylation of CPI-17 and reduction of myosin phosphatase activity. With the reasonable assumption that the elevated levels of cGMP [11] activated PKG, it is proposed that these results indicate that CPI-17 can be phosphorylated in vivo by PKG at the inhibitory site. It is not known if an increase in [cAMP] would produce similar results. It is possible, therefore, that the response to increased [cGMP] and possibly [cAMP] is to promote an initial relaxation and reduction of force in smooth muscle and subsequently via the phosphorylation of CPI-17 to moderate phosphatase activity. The molecular basis for the temporal and selective phosphorylation of CPI-17 by PKG is not known, but could involve other components such as phosphorylated CPI-17 phosphatase and changes in intracellular Ca2þ levels. The roles of PHI-1 and KEPI in cell function remain to be determined but based on the above results it is unlikely that cyclic nucleotide-dependent phosphorylation increases their inhibitory properties. ILK was shown to phosphorylate both CPI-17 and PHI-1 and to increase the inhibitory potency of these proteins on myosin phosphatase inducing Ca2þ -sensitization of smooth muscle contraction in permeabilized tissue [21]. ILK may also modulate smooth muscle contraction by direct phosphorylation of myosin [22] and inhibitory phosphorylation of MYPT1 [29,30]. Our present data suggest that phosphorylation by ILK results in a dramatic increase in the inhibitory potency of KEPI on both PP1c and MPH. It is likely that phosphorylation of KEPI by ILK may represent an alternative pathway for the regulation of myosin phosphorylation in tissues where KEPI is expressed. KEPI is predominant in brain, skeletal muscle, and heart [10] and these tissues also include MPH [13]. It remains to be elucidated if phosphorylated KEPI also inhibits other PP1 holoenzymes. Although there is precedent for the in vivo phosphorylation of CPI-17, it is necessary to monitor the phosphorylation status of the other inhibitors under various conditions in intact cells. The signaling pathways leading to the activation/
inhibition of ILK are not known, therefore correlations between the phosphorylation of these inhibitory proteins (CPI-17, PHI-1, and KEPI) and the activity of ILK in cells are difficult to determine. Nevertheless, our present in vitro data support the conclusion that ILK may represent an enzyme central to the regulation of PP1 holoenzymes in a variety of cell types.
Acknowledgments This work was supported by OTKA T043296 (from the Hungarian Science Research Fund), ETT (from the Ministry of Health of Hungary), and OM FKFP 0616/2000 (from the Ministry of Education of Hungary) grants (to F.E.), HL 23615 Grant from the National Institutes of Health (to D.J.H.), grants from the Canadian Institutes of Health Research (to M.P.W.), United States Public Health Service NIGMS Grant GM56362 and NCI Grant CA40042 from the National Institutes of Health (to D.L.B.), and a Scientist Development Grant from the American Heart Association National Center (to M.E.). M.P.W. is an Alberta Heritage Foundation for Medical Research Medical Scientist and recipient of a Canada Research Chair (Tier I) in Biochemistry. The authors are grateful to Dr. Andrea Muranyi (University of Arizona) for the preparation of MPH, to Csilla Dudas (University of Arizona) for help with the manuscript and to Dr. David P. Wilson (University of Calgary) for helpful discussions.
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Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinasesq Ferenc Erd} odi,a,* Enik} o Kiss,a Michael P. Walsh,b Bjarki Stefansson,c Jing Ti Deng,b Masumi Eto,c David L. Brautigan,c and David J. Hartshorned a
Department of Medical Chemistry, Medical and Health Science Center, University of Debrecen, H-4012 Debrecen, Bem t er 18/B, Hungary b Smooth Muscle Research Group and Canadian Institutes of Health Research Group in Regulation of Vascular Contractility, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alta., Canada T2N 4N1 c Center for Cell Signaling, University of Virginia School of Medicine, Box 800577-MSB 7225, Charlottesville, VA 22908, USA d Muscle Biology Group, University of Arizona, Shantz Bldg. 601, Tucson, AZ 85721, USA Received 9 May 2003
Abstract Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC50 values of 1–8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC50 ¼ 0:1 nM) and MPH (IC50 ¼ 8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: C-kinase-enhanced (potentiated) phosphatase inhibitors (CPI-17 and KEPI); Myosin phosphatase; Protein kinase A; Protein kinase G
Protein phosphorylation is a widespread regulatory mechanism and phosphorylation levels are a reflection of the balance between kinase and phosphatase activities, which can themselves be subjected to regulation. Cellular protein phosphatase-1 (PP1) activity is controlled by interaction with various targeting subunits
q
Abbreviations: CPI-17, C-kinase potentiated 17 kDa inhibitor of type-1 protein phosphatase; ILK, integrin-linked kinase; KEPI, kinase-enhanced protein phosphatase type-1 inhibitor; LC20, 20 kDa light chain of smooth muscle myosin II; MPH, myosin phosphatase holoenzyme; MYPT1, myosin phosphatase target subunit; PHI, phosphatase holoenzyme inhibitor; PKA, cAMP-dependent protein kinase (protein kinase A); PKG, cGMP-dependent protein kinase (protein kinase G); PP1, type-1 protein phosphatase; PP1c, catalytic subunit of PP1; ROK, Rho-associated kinase; I-1, inhibitor-1; I-2, inhibitor-2; DARPP-32; dopamine and cAMP regulated 32 kDa phosphoprotein. * Corresponding author. Fax: +36-52-412566. E-mail address: [email protected] (F. Erd} odi).
and PP1 inhibitory proteins (reviews [1–4]). PP1 inhibitory proteins have been known for decades. The earlier discoveries included inhibitors of type-1 protein phosphatase (PP1): inhibitor 1 (I-1), inhibitor-2 (I-2), and DARPP-32. I-1 and DARPP-32 are phosphorylated by cAMP-dependent kinase (PKA), which markedly increases the inhibitory potency. More recently, a PP1 inhibitor, called CPI-17 (a 17 kDa PKC-potentiated inhibitory protein of PP1), was discovered in porcine aorta [5]. CPI-17 is exclusively expressed in smooth muscles and in brain [6–8]. It was shown that phosphorylation of T38 by PKC increased inhibitory potency over 1000-fold [6]. Subsequently, orthologues of CPI-17, called PHI-1 (phosphatase holoenzyme inhibitor-1) [9] and KEPI (kinase-enhanced protein phosphatase-1 inhibitor) [10], were identified in mammalian tissues. PHI-1 is ubiquitously expressed among tissues, whereas KEPI is predominantly expressed in brain, heart, and muscle. Sequences around the inhibitory sites
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00976-8
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of phosphorylation in these proteins are conserved: 35 ARVTV40 K for CPI-17, 54 GKVTV59 K for PHI-1, and 69 GKVTV74 K for KEPI, i.e., consensus sequences for PKC. Indeed, PHI-1 and KEPI are also activated by phosphorylation with purified PKC [6,9,10]. Recently, in a study carried out with intact carotid artery it was found that prolonged exposure to an NO donor resulted in phosphorylation of CPI-17, suggesting involvement of PKG and/or PKA in regulation of the CPI-17 family [11]. One of the systems in which there is considerable interest in signaling pathways involved in regulation of phosphatase activity is smooth muscle. Phosphorylation of the myosin regulatory light chains (LC20) is an important regulatory mechanism and activation or inhibition of phosphatase leads to decreased or increased levels of myosin phosphorylation, respectively. In vascular muscle it is thought that agonist stimulation leads to inhibition of phosphatase and increased contraction at sub-optimal Ca2þ concentrations, whereas elevated cyclic nucleotides promote an opposite effect [12]. The myosin phosphatase holoenzyme (MPH) is composed of three subunits: a catalytic subunit PP1c (d isoform) and two non-catalytic subunits of approximately 18–20 kDa (M20) and 106–110 kDa [13]. The latter is termed myosin phosphatase target subunit, MYPT1. Two mechanisms have been proposed for inhibition of MPH: one via phosphorylation of MYPT1 by, e.g., ROK [14], and the other by phosphorylated CPI-17 [15]. Previously it was shown that I-1 and I-2 were not effective inhibitors of MPH [16] but that CPI-17 was effective (IC50 of 12 nM) [17]. PHI-1 can also inhibit MPH, although the potency of PHI-1 (IC50 of 30 nM) is lower than that of CPI-17 [9]. The effect of KEPI on MPH has not yet been determined. In addition to PKC, several kinases have been shown to phosphorylate CPI-17 and potentiate inhibition [18– 21]. ILK was shown to phosphorylate both CPI-17 and PHI-1 [21]. Phosphorylated CPI-17 and PHI-1 exhibited increased phosphatase inhibitory potency and induced Ca2þ -sensitization of contraction of Triton X-100 demembranated rat-tail arterial smooth muscle. The influence of phosphorylation by different kinases (other than PKC) has not been studied with KEPI. Our objectives were to investigate possible phosphorylation of MPH inhibitors (focusing on CPI-17 and KEPI) by PKA and PKG, as well as ILK. To document effects of phosphorylation on phosphatase activity, we used native PP1c and MPH purified from skeletal and smooth muscles, respectively.
Materials and methods Materials. Chemicals and vendors were as follows: [c-32 P]ATP, Perkin–Elmer Life Sciences (Boston, MA, USA); ATP, Sigma (St. Louis, MO, USA); adenosine 50 -[c-thio]triphosphate (ATPcS), Cal-
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biochem (San Diego, CA, USA); nitrocellulose membrane (0.45 lm pore size) and enhanced chemiluminescence (ECL) reagent kit, Amersham Pharmacia Biotech (Piscataway, NJ, USA); and nitrocellulose membrane (0.20 lm pore size), Bio-Rad Laboratories (Hercules, CA, USA). All other chemicals used were the highest grade commercially available. Proteins. ILK [22] and MPH [23] were purified from chicken and turkey gizzard, respectively. Purification procedures for the following proteins are given in [24]: PP1 catalytic subunit (PP1c) from rabbit skeletal muscle, recombinant hexahistidine-tagged PP1c (d isoform), LC20 of gizzard myosin, and 32 P-labeled LC20. Hexahistidine-tagged wild-type (WT) and mutant CPI-17 (CPI-17WT , CPI-17T38A , and CPI1735–147 ) [25] and hexahistidine-tagged PHI-1 [9] were expressed and purified as described previously. Polyclonal antibodies that recognize phosphorylated T38 in CPI-17 (anti-CPI-17pT38 ) [26] and T57 in PHI-1 (anti-PHI-1pT57 ) [21] were used as described previously. The cDNA fragment of human KEPI was amplified by PCR methods using an IMAGE clone No. 4877511 as a template, purchased from Incyte Genomics (CA, USA), and inserted into pET30 vector as described previously [6]. Mutation of T73 of KEPI to A was performed using the QuickChange site-directed mutagenesis kit (Stratagene, CA, USA). Hexahistidine-tagged wild-type and mutant KEPI (KEPIWT and KEPIT73A ) were prepared from bacterial lysates as described previously [6]. PKA inhibitor peptide and horseradish peroxidase-coupled goat anti-rabbit immunoglobulin were from Sigma (St. Louis, MO, USA), PKG was from Calbiochem (San Diego, CA, USA), PKA and ROK (ROKa/ROCKII) were from Upstate Biotechnology (Lake Placid, NY, USA). Phosphorylation of PP1 inhibitory proteins. Phosphorylation of wild-type or mutant CPI-17, PHI-1 or KEPI (10 lM of each) was carried out in an assay buffer containing 30 mM Tris–HCl (pH 7.5), 85 mM KCl, 10 mM EGTA, 10 mM dithiothreitol, 1% Tween 80, 1 lM microcystin-LR, 10 mM MgCl2 , and 0.2 mM [c-32 P]ATP (100– 500 cpm/pmol) in the presence of 2 lg/ml PKA or PKG, or 20% (v/v) of ILK in a total volume of 50 or 100 ll. The reaction was started by addition of [c-32 P]ATP and 5 or 10 ll aliquots were removed and spotted on P81 paper discs. The filter papers were treated as described [27] and radioactivity was quantified in a scintillation counter. The stoichiometry of phosphorylation was calculated from the amount of 32 P incorporated into the proteins. Phosphorylation was also verified by autoradiography after separation of the proteins by SDS–PAGE. The gels were dried and exposed to X-ray film. Phosphorylation was also carried out in the absence of microcystin-LR and with replacement of [c-32 P]ATP with 0.2 mM ATPcS to thiophosphorylate the proteins. The thiophosphorylated proteins were used to assay the influence of phosphorylation of CPI-17 or KEPI on the activity of PP1c or MPH. Thiophosphorylated CPI-17 and KEPI were also used to assess inhibitory phosphorylation of T38 in CPI-17 or T73 in KEPI. CPI-17 and KEPI (or their phosphorylation site mutants T38A or T73A) were subjected to SDS–PAGE, and the proteins were transferred to nitrocellulose membrane and detected with phosphospecific antibodies: anti-CPI-17pT38 or anti-PHI-1pT57 . Anti-PHI-1pT57 , but not anti-CPI-17pT38 , recognized phosphorylated T73 in KEPI. Phosphatase assays. Phosphatase activity of skeletal muscle PP1c (0.5 nM), recombinant His-PP1cd (0.5 nM) or MPH was determined with 5 lM 32 P-labeled LC20 substrate as described previously [24]. Effectors (non-phosphorylated and phosphorylated CPI-17 and KEPI proteins) were preincubated with the phosphatase for 5 min, and then the reaction was initiated by addition of the substrate.
Results and discussion Phosphorylation of CPI-17 and two mutants by PKA is shown in Fig. 1A. Wild-type CPI-17 was phosphory-
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Fig. 1. Phosphorylation of CPI-17 by PKA and PKG. (A) PKA: (s) CPI-17WT ; (M) CPI-17T38A ; () CPI-1735–147 ; (d) CPI-17WT plus 10 lM PKA inhibitor peptide. (B) PKG: (s) CPI-17WT ; (M) CPI-17T38A ; () CPI-1735–147 . Data are means SE (n ¼ 3–6). (C) Autoradiograms (AU) and Western blots (WB) with anti-CPI-17pT38 of CPI-17WT and CPI-17T38A phosphorylated by PKA or PKG.
lated to 0.5 mol P/mol protein and this phosphorylation was inhibited completely by the peptide inhibitor of PKA (Fig. 1A), confirming phosphorylation by PKA rather than a contaminating kinase. Mutation of the inhibitory site of phosphorylation in CPI-17T38A resulted in a reduced level of phosphorylation by PKA (0.2 mol P/mol) but, surprisingly, did not eliminate phosphorylation (Fig. 1A), indicating that CPI-17 has more than one site of phosphorylation by PKA. A truncation mutant of CPI-17 lacking the first 34 residues (CPI1735–147 ) was phosphorylated to <0.1 mol P/mol, suggesting that phosphorylation at T38 was reduced by elimination of residues N-terminal to the consensus sequence. Earlier it was shown that ROK phosphorylated
CPI-17 at T38 [18]. In addition to T38, S12 of CPI-17 is phosphorylated by PKC [5] and MYPT1 kinase [20] at substoichiometric levels, suggesting that phosphorylation at T38 promoted phosphorylation at S12. Positive cooperative phosphorylation was not observed with sequential phosphorylation of CPI-17, initially by ROK followed by PKA (data not shown), in contrast to the expected T38 and S12 phosphorylation in CPI-17 by MYPT1 kinase [20]. Phosphorylation of CPI-17 by PKG is shown in Fig. 1B. Compared to PKA the phosphorylation rate is faster and the stoichiometry of phosphorylation higher (0.75 mol P/mol). Another difference relative to PKA is that the two mutants, CPI-17T38A and CPI-1735–147 , showed very low levels of phosphorylation with PKG (0.04 and 0.03 mol P/mol, respectively). Autoradiography (Fig. 1C) confirmed that wild-type CPI-17 was phosphorylated by both PKA and PKG and that CPI17T38A was phosphorylated only by PKA. Use of an antibody specific for phosphorylated T38 confirmed that both PKA and PKG phosphorylated the inhibitory site of CPI-17 (Fig. 1C). CPI-17 phosphorylated by either PKA or PKG inhibited the activity of rabbit skeletal muscle PP1c with IC50 values of 4.0 and 1.4 nM, respectively (Fig. 2). The lower potency of inhibition for CPI-17 phosphorylated by PKA probably reflects the lower level of phosphorylation at T38 (0.5 mol P/mol) compared to PKG-phosphorylated CPI-17 (0.75 mol P/mol). Nonphosphorylated wild-type CPI-17 and CPI-17T38A phosphorylated by PKA did not inhibit native PP1c (Fig. 2). It is interesting that MPH was also inhibited by CPI-17 phosphorylated by PKG, but with lower potency (IC50 ¼ 8:2 nM) compared to isolated PP1c (Fig. 2). These results lead to an unexpected conclusion, namely that PKA and PKG can both phosphorylate CPI-17 at the inhibitory site and effect inhibition of isolated PP1c and MPH. In these respects
Fig. 2. Inhibition of phosphatase activity by non-phosphorylated and thiophosphorylated CPI-17. PP1c with: (}) non-phosphorylated CPI17WT ; () CPI-17WT thiophosphorylated by PKG; (M) CPI-17WT thiophosphorylated by PKA; and (O) CPI-17T38A thiophosphorylated by PKA. Myosin phosphatase holoenzyme with: (j) CPI-17WT thiophosphorylated by PKG. Data are means SE (n ¼ 2–4).
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PKA and PKG are similar to several other kinases [6,18–21]. The sequence similarity around the inhibitory phosphorylation sites of CPI-17, PHI-1, and KEPI prompted a comparative study of phosphorylation of these proteins by PKA and PKG. With PHI-1 as substrate, very low levels of phosphorylation (<0.1 mol P/mol) by PKA or PKG were obtained (data not shown). Results with wild-type KEPI and a phosphorylation site mutant, KEPIT73A , are shown in Fig. 3A. Phosphorylation of wild-type and mutant KEPI by PKA was also observed and, surprisingly, the phosphorylation site mutant, KEPIT73A , was a slightly more effective substrate. Thus, it is unlikely that T73 in KEPI is a major site of phosphorylation by PKA. This was also confirmed by Western blots with anti-PHI-1pT57 (Fig. 3B): wild-type KEPI phosphorylated by PKA gave a faint signal indicating a low level of phosphorylation at T73. An alternative site of phosphorylation by PKA in KEPI is S157, as suggested previously [10]; this site fits the PKA consensus sequence (RKLSP). PKG did not phosphorylate either the wild type or mutant KEPI (Fig. 3A). Thus, unlike T38 of CPI-17, cyclic nucleotide-dependent kinases fail to phosphorylate T57 of PHI-1 or T73 of KEPI, despite conserved amino acid sequences. On the other hand, ILK phosphorylated wild-type KEPI (KEPIWT ) but not KEPIT73A (Fig. 3A). Because the sequence of the phospho-peptide used as antigen to
Fig. 3. Phosphorylation of KEPI by ILK and PKA, but not PKG. (A) ILK: (s) KEPIWT ; (d) KEPIT73A . PKA: (M) KEPIWT ; (O) KEPIT73A . PKG: () KEPIWT ; (j) KEPIT73A . Data are means SE (n ¼ 3). (B) Coomassie blue-stained SDS gel, autoradiograms (AU), and Western blots (WB) with anti-PHI-1pT57 of KEPIWT and KEPIT73A phosphorylated by ILK or PKA.
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produce anti-PHI-1pT57 is identical to the corresponding sequence in KEPI, anti-PHI-1pT57 cross-reacted with wild-type KEPI phosphorylated by PKC (data not shown). The antibody also bound to KEPIWT but not the T73A mutant phosphorylated by ILK on Western blots (Fig. 3B). Phosphoamino acid analysis showed phosphorylation at both S and T by PKC, whereas ILK specifically phosphorylated T in KEPI (data not shown). The results indicate specific phosphorylation of KEPI at T73 by ILK. The effects of non-phosphorylated and thiophosphorylated wild-type and mutant KEPI were assayed using native PP1c (from skeletal muscle), recombinant PPlcd (from bacterial expression), and MPH. Previously it was found that KEPI phosphorylated by PKC inhibited recombinant PP1c (using phosphorylase a as substrate) with an IC50 of 2.7 nM [10]. In Fig. 4 it is shown that unphosphorylated KEPI had an IC50 of 1.8 lM. Wildtype KEPI phosphorylated by ILK inhibited each PP1c preparation with IC50 values of 3.3 nM for recombinant PP1cd and 0.1 nM for native PP1c. The point to emphasize is that recombinant PP1cd is less sensitive to inhibition by KEPI (similar results were obtained with I-1 [28]). Furthermore, it is noteworthy that phosphoKEPI potently inhibits MPH with an IC50 value of 8.6 nM. Thus, in addition to CPI-17 and PHI-1, KEPI is a third inhibitor that can suppress the myosin phosphatase holoenzyme, although the holoenzyme is 80fold less sensitive to inhibition than isolated PP1c. Wild-type KEPI phosphorylated by PKA gave an IC50 of 17 nM (with rabbit PP1c) whereas KEPIT73A phosphorylated by PKA was similar to unphosphorylated KEPI (IC50 0:6 lM), suggesting that the putative S157 site is not inhibitory and the inhibition observed with PKA-phosphorylated KEPI is due to fractional phosphorylation at T73.
Fig. 4. Inhibition of phosphatase activity by nonphosphorylated and thiophosphorylated KEPI. PP1c with: (}) non-phosphorylated KEPIWT ; () non-phosphorylated KEPIT73A ; (s) KEPIWT thiophosphorylated by ILK; (M) KEPIWT thiophosphorylated by PKA; and (O) KEPIT73A thiophosphorylated by PKA. Recombinant PP1cd with: () KEPIWT thiophosphorylated by ILK. Myosin phosphatase holoenzyme with: (d) KEPIWT thiophosphorylated by ILK. Data are means SE (n ¼ 2–3).
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Kitazawa et al. [26] showed in femoral artery preparations that CPI-17 was phosphorylated in response to several stimulants including histamine (intact strips) and GTPcS (a-toxin permeabilized strips). Inhibitors of ROK and PKC both reduced contraction and CPI-17 phosphorylation, suggesting the involvement of at least two signal transduction pathways. Thus, under these conditions the phosphorylation of CPI-17 favored an increased contractile response, as expected. Other results consistent with the proposed role of CPI-17 relative to myosin phosphatase activity are that addition of nitroprusside (an NO donor) to histamine-contracted carotid artery resulted in relaxation via decreased CPI-17 phosphorylation and increased myosin phosphatase activity [11]. However, an intriguing observation was that prolonged exposure to nitroprusside resulted in phosphorylation of CPI-17 and reduction of myosin phosphatase activity. With the reasonable assumption that the elevated levels of cGMP [11] activated PKG, it is proposed that these results indicate that CPI-17 can be phosphorylated in vivo by PKG at the inhibitory site. It is not known if an increase in [cAMP] would produce similar results. It is possible, therefore, that the response to increased [cGMP] and possibly [cAMP] is to promote an initial relaxation and reduction of force in smooth muscle and subsequently via the phosphorylation of CPI-17 to moderate phosphatase activity. The molecular basis for the temporal and selective phosphorylation of CPI-17 by PKG is not known, but could involve other components such as phosphorylated CPI-17 phosphatase and changes in intracellular Ca2þ levels. The roles of PHI-1 and KEPI in cell function remain to be determined but based on the above results it is unlikely that cyclic nucleotide-dependent phosphorylation increases their inhibitory properties. ILK was shown to phosphorylate both CPI-17 and PHI-1 and to increase the inhibitory potency of these proteins on myosin phosphatase inducing Ca2þ -sensitization of smooth muscle contraction in permeabilized tissue [21]. ILK may also modulate smooth muscle contraction by direct phosphorylation of myosin [22] and inhibitory phosphorylation of MYPT1 [29,30]. Our present data suggest that phosphorylation by ILK results in a dramatic increase in the inhibitory potency of KEPI on both PP1c and MPH. It is likely that phosphorylation of KEPI by ILK may represent an alternative pathway for the regulation of myosin phosphorylation in tissues where KEPI is expressed. KEPI is predominant in brain, skeletal muscle, and heart [10] and these tissues also include MPH [13]. It remains to be elucidated if phosphorylated KEPI also inhibits other PP1 holoenzymes. Although there is precedent for the in vivo phosphorylation of CPI-17, it is necessary to monitor the phosphorylation status of the other inhibitors under various conditions in intact cells. The signaling pathways leading to the activation/
inhibition of ILK are not known, therefore correlations between the phosphorylation of these inhibitory proteins (CPI-17, PHI-1, and KEPI) and the activity of ILK in cells are difficult to determine. Nevertheless, our present in vitro data support the conclusion that ILK may represent an enzyme central to the regulation of PP1 holoenzymes in a variety of cell types.
Acknowledgments This work was supported by OTKA T043296 (from the Hungarian Science Research Fund), ETT (from the Ministry of Health of Hungary), and OM FKFP 0616/2000 (from the Ministry of Education of Hungary) grants (to F.E.), HL 23615 Grant from the National Institutes of Health (to D.J.H.), grants from the Canadian Institutes of Health Research (to M.P.W.), United States Public Health Service NIGMS Grant GM56362 and NCI Grant CA40042 from the National Institutes of Health (to D.L.B.), and a Scientist Development Grant from the American Heart Association National Center (to M.E.). M.P.W. is an Alberta Heritage Foundation for Medical Research Medical Scientist and recipient of a Canada Research Chair (Tier I) in Biochemistry. The authors are grateful to Dr. Andrea Muranyi (University of Arizona) for the preparation of MPH, to Csilla Dudas (University of Arizona) for help with the manuscript and to Dr. David P. Wilson (University of Calgary) for helpful discussions.
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