Báo cáo Y học: Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinase docx

Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinase P. V. Sathyanarayanan and B. W. Poovaiah 1 Laboratory of Plant Molecular Biology and Physiology, Department of Horticulture, Washington State University, Pullman, WA, USA Chimeric calcium/calmodulin dependent protein kinase (CCaMK) is characterized by the presence of a visinin-like Ca 2+ -binding domain unlike other known calmodulin- dependent kinases. Ca 2+ -Binding to the visinin-like domain leads to autophosphorylation and changes in the affinity for calmodulin [Sathyanarayanan P.V., Cremo C.R. & Poovaiah B.W. (2000) J. Biol. Chem. 275, 30417–30422]. Here, we report that the Ca 2+ -stimulated autophosphory- lation of CCaMK results in time-dependent loss of enzyme activity. This time-dependent loss of activity or self-inacti- vation due to autophosphorylation is also dependent on reaction pH and ATP concentration. Inactivation of the enzyme resulted in the formation of a sedimentable enzyme due to self-association. Specifically, autophosphorylation in thepresenceof200l M ATP at pH 7.5 resulted in the for- mation of a sedimentable enzyme with a 33% loss in enzyme activity. Under similar conditions at pH 6.5, the enzyme lost 67% of its activity and at pH 8.5, 84% enzyme activity was lost. Furthermore, autophosphorylation at either acidic or alkaline reaction pH lead to the formation of a sedimentable enzyme. Transmission electron microscopic studies on autophosphorylated kinase revealed particles that clustered into branched complexes. The autophosphorylation of wild- type kinase in the presence of AMP-PNP (an unhydrolyz- able ATP analog) or the autophosphorylation-site mutant, T267A, did not show formation of branched complexes under the electron microscope. Autophosphorylation- dependent self-inactivation may be a mechanism of modu- lating the signal transduction pathway mediated by CCaMK. Keywords: self-inactivation; self association; autophospho- rylation; Ca 2+ ; chimeric calcium/calmodulin dependent protein kinase. Ca 2+ regulates a large number of physiological and developmental processes [1]. The effects of Ca 2+ are so wide spread that it becomes difficult to pinpoint specific mechanisms of Ca 2+ signal transduction. Ca 2+ signaling is orchestrated through several calcium binding proteins such as calmodulin, ion channels, Ca 2+ -dependent protein kinases and Ca 2+ /calmodulin dependent protein kinases [2,3]. A large number of plant Ca 2+ -dependent protein kinases (CDPK) have been reported [4–7]. These kinases require Ca 2+ for autophosphorylation and substrate phos- phorylation [4–7]. However, there is only limited informa- tion available about the Ca 2+ /CaM-dependent protein kinases in plants [7,8]. Chimeric calcium calmodulin dependent protein kinase (CCaMK) has been cloned from lily anthers [9]. CCaMK is stage-specifically expressed in tapetal cells and pollen mother cells of anthers during male gametophyte develop- ment [10]. CCaMK is characterized by a serine-threonine kinase domain, an autoinhibitory domain overlapping with calmodulin binding domain and a C-terminal visinin-like domain with three calcium-binding sites [9]. Visinin-like proteins are high affinity Ca 2+ -binding proteins and function as Ca 2+ sensors in neurons [30–33] 2 . The calmod- ulin-binding domain of CCaMK is very similar to CaM kinase II [11,12]. Ca 2+ binding to the C-terminal visinin-like domain leads to autophosphorylation of the kinase [8,12]. Unlike CDPKs, the substrate phosphorylation by CCaMK requires both Ca 2+ and CaM [7,8,11]. The interaction between the CCaMK and CaM is modulated by the Ca 2+ - stimulated autophosphorylation [8]. CaM-dependent pro- tein kinases reported from invertebrates and vertebrates require Ca 2+ /CaM for autophosphorylation [13]. In the present study, we report a new property of CCaMK associated with Ca 2+ -stimulated autophosphorylation. Ca 2+ -stimulated autophosphorylation of CCaMK resulted in a time-dependent loss of kinase activity. This property, described as self-inactivation, is sensitive to reaction pH and ATP concentration. Furthermore, the autophosphorylation- dependent inactivation leads to the formation of a sediment- able enzyme. When observed under transmission electron microscope, the autophosphorylated kinase appeared as particles that are clustered into branched complexes. EXPERIMENTAL PROCEDURES Materials AMP-PNP and ATP were purchased from Sigma Chemical Co. and [c- 32 P]ATP (3000 CiÆmmol )1 ) from Dupont Corp. Correspondence to B. W. Poovaiah, Laboratory of Plant Molecular Biology and Physiology, Department of Horticulture, Washington State University, Pullman, WA 99164-6414, USA. Fax: + 1 509 335 8690, Tel.: + 1 509 335 2487, E-mail: poovaiah@wsu.edu Abbreviations:CaM,calmodulin;CCaMK,chimericcalcium/ calmodulin-dependent protein kinase; TEM, transmission electron microscopy; AMP-PNP, adenosine 5¢-(b,-imido)triphosphate; CaMK II, calcium/calmodulin-dependent protein kinase II. (Received 24 October 2001, revised 20 March 2002, accepted 22 March 2002) Eur. J. Biochem. 269, 2457–2463 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02904.x Tabbed-copper grids (400 mesh) and polystyrene sizing beads (93 and 262 nm) were obtained from Ted Pella (Redding, CA, USA) 3 . Purification of CCaMK CCaMK cDNA from Lily (Lilium longiflorum Thunb cv. Nellie white) and phosphorylation mutant T267A were cloned into the pET3b expression vector (Novagen. Inc.) and expressed in E. coli. These proteins were purified as described previously [11,12]. Autophosphorylation assays The autophosphorylation assay of CCaMK and mutant protein were carried out [11] for 10 min at 30 °Cinthe presence of 50 m M Hepes, pH 7.5, containing 10 m M magnesium acetate, 1 m M dithiothreitol, 1 m M [c- 32 P]ATP and either 2.5 m M EGTA or 0.2 m M CaCl 2 . For the autophosphorylation under different pH condi- tions, 50 m M Hepes, at pH 6.5 and 8.5, were used under the same conditions as described above. Aliquots (10 lL, 200 ng enzyme) were collected at 0, 2, 6 min and diluted to 100 lLoficecold50m M Hepes, pH 7.5 with 10 m M EDTA. This terminated further autophosphorylation of the enzyme. Samples were then centrifuged for 30 min at 16 000 g at 4 °C. The supernatant and pellet were separated and suspended in SDS loading buffer (10% glycerol, 15 m M dithiothreitol, 2.3% SDS and 62.5 m M Tris, pH 6.8) for SDS/PAGE analysis [14]. [ 32 P]PO 4 incorporation was measured by excising the protein bands from the gel and counting using a scintillation counter. Kinase assays The autophosphorylation-induced changes in the kinase activity was studied using a second stage assay. An aliquot of autophosphorylated enzyme (2.5 lL, 100 ng of enzyme) from the first stage reaction was added to the second stage reaction mix (final volume, 20 lL) consisting of 50 m M Hepes, pH 7.5, containing 10 m M magnesium acetate, 1 m M dithiothreitol, 1 m M [c- 32 P]ATP, 0.2 m M CaCl 2 ,0.5l M calmodulin, 100 l M Histone II AS. The second stage reaction mix was preincubated at 30 °C before the addition of enzyme. The substrate phosphory- lation was allowed to proceed for 10 min. The reaction was stopped by the addition of SDS loading buffer. The samples were analyzed on a 12% SDS/polyacrylamide gel and 32 PO 4 incorporation was measured by excising protein bands from the gel and counting using a scintillation counter. Transmission electron microscopy All the solutions used for transmission electron microscopy were filtered using 0.2-lm filters to remove any impurities. CCaMK was autophosphorylated as described above and the reaction was terminated by the addition of EDTA (to a final concentration of 50 m M ) and the sample was kept in ice. The autophosphorylated and unphosphorylated kinase samples were deposited onto the carbon coated Formvar grids by the floating method, as described previously [15]. Drops (50 lL) of the kinase sample and other solutions were placed on Parafilm stretched over a top of a Petri dish. A grid was placed on a drop of the autophospho- rylated kinase for 45 s and then placed on a drop of uranyl acetate stain (2% uranyl acetate in 25% methanol) for 1 min. The grid was subsequently placed on a drop of distilled water for 45 s and air dried. The polystyrene sizing beads (93 and 262 nm diameter) were applied to the grids following the procedures outlined above for size reference. Transmission electron microscopy was performed using conventional procedures on a JOEL-100CX operating at 80 kV. RESULTS Autophosphorylation and kinase activity of CCaMK Autophosphorylation of CCaMK was associated with a time dependent loss of the Ca 2+ /CaM-dependent enzyme activity at pH 6.5, 7.5 and 8.5 (Fig. 1). Activity meas- urements of enzymes phosphorylated at these different pH conditions indicated that the enzyme activity decrea- sed overtime with either a decrease or increase in the reaction pH (Fig. 1). Autophosphorylation of CCaMK at pH 6.5 resulted in 67% loss of enzyme activity in 2 min. While at pH 7.5, autophosphorylation-dependent loss was 33% and at pH 8.5, CCaMK lost about 84% of enzyme activity. A time-course experiment was conduc- ted to study the kinetics of inactivation (Fig. 2, Table 1). The results in Fig. 2 indicate that the loss of enzyme activity follow an exponential decay curve (Fig. 2). CCaMK lost 28% activity at pH 7.5, 54% activity at pH 6.5 and 72% activity at pH 8.5 in 30 s after autophosphorylation. Fig. 1. Self-inactivation of CCaMK during autophosphorylation. Autophosphorylation was initiated by the addition of 500 ng CCaMK to the reaction mixture (see Experimental procedures) at different pH conditions. Aliquots of the autophosphorylation mix were collected at the indicated time points and Ca 2+ /CaM dependent activity was determined as described. Each bar represents mean of four inde- pendent measurements. 2458 P. V. Sathyanarayanan and B. W. Poovaiah (Eur. J. Biochem. 269) Ó FEBS 2002 Autophosphorylation results in the formation of sedimentable enzyme At 0, 2 and 6 min of autophosphorylation, an aliquot of the reaction mixture at different pH and ATP concentration were subjected to centrifugation for 30 min at 16 000 g,to determine whether any sedimentable enzyme is formed under these conditions. Figure 3 shows the formation of the sedimentable enzyme after 2 min of autophosphorylation. Autophosphorylation at different ATP concentrations (200 l M and 1 m M ; Fig. 3) shows that at higher concentra- tions of ATP, less sedimentable enzyme is formed. Transmission electron microscopy studies of autophosphorylated CCaMK The formation of sedimentable enzyme was visualized using TEM. CCaMK phosphorylated at 200 l M ATP for 5 min produced distinct uranyl acetate staining structures (Fig. 4A,B). These particles appeared to interconnect and associate to form branched structures. Figure 4C shows the electron micrograph of an autophosphorylation deficient site directed mutant (T267A) of CCaMK that did not form branched complexes in the presence of 200 l M ATP for 5 min. In addition, autophosphorylation of T267A for 10 min did not show any branched complexes under TEM. Autophosphorylation in the presence of adenosine 5¢-(b- imido) triphosphate (AMP-PNP) 4 , an unhydrolyzable ana- logue of ATP also did not produce uranyl acetate staining structures under TEM (Fig. 4D). DISCUSSION It was shown that unlike other reported CaM-dependent kinases, CCaMK requires Ca 2+ for autophosphorylation and Ca 2+ /CaM is required for substrate phosphorylation [7–9,11,12]. The substrate phosphorylation depends on the extent of Ca 2+ stimulated autophosphorylation [9,12]. Ca 2+ binding to the C-terminal visinin-like domain results is phosphorylation of threonine 267 [12]. In this study, we report autophosphorylation-dependent inactivation of CCaMK. Autophosphorylation of CCaMK resulted in time-dependent loss of enzyme activity (Figs 1 and 2; Table 1) and formation of a sedimentable enzyme (Fig. 3). Autophosphorylation-dependent loss of enzyme activity was influenced by both acidic and alkaline reaction pH. Autophosphorylation at pH 8.5 produced a robust inacti- vation leading to a loss of 72% enzyme activity in 30 s (Fig. 2, Table 1). Among the three different pH conditions tested, loss of enzyme activity was lowest at pH 7.5 (Fig. 1). Time-dependent decrease in the activity of autophosphor- ylated CaMK II has also been reported [16–24]. Auto- phosphorylation of CaMK II at pH 7.5 resulted in 15% decrease in activity at 2 min [24]. CCaMK lost about 33% of enzyme activity in 2 min, indicating that it is more sensitive to autophosphorylation-dependent inactivation. At pH 6.5, there was about 40% loss of enzyme activity in 2 min for CaMK II [24] and at this pH, CCaMK lost 67% of enzyme activity. These results suggest that the enzyme inactivation is dependent on the duration of autophospho- rylation and reaction pH. The fit to the time-dependent kinetics showed (Fig. 2) that the inactivation followed exponential decay [R values: Table 1. Time-dependent inactivation of kinase activity of CCaMK due to autophosphorylation. CCaMK was autophosphorylated as described in Experimental procedures, and an aliquot (200 ng of CCaMK) was added at indicated time points to a second stage reaction mixture containing 200 l M Histone II AS, as substrate. The phosphorylation was allowed for 10 min and [ 32 P]PO 4 incorporation into the Histone II AS was measured by excising protein bands from the gel and counting using a scintillation counter. The phosphorylation (c.p.m.) represents mean of three measurements. Time (min) pH 6.5 pH 7.5 pH 8.5 Phosphorylation (c.p.m.) % Initial activity Phosphorylation (c.p.m.) % Initial activity Phosphorylation (c.p.m.) % Initial activity 0 84010 100 110998 100 48267 100 0.5 38259 46 80140 72 13286 28 2 28315 34 74543 67 7789 16 4 24066 29 69208 62 4440 9 6 22410 27 59127 53 3997 8 8 22671 27 60190 54 4293 9 Fig. 2. Time course of inactivation of CCaMK during autophosphory- lation. Autophosphorylation was performed at different reaction pH (data points m,pH7.5;d,pH6.5;j, pH 8.5) as described. Aliquots of the autophosphorylation mix were collected at the indicated time points and Ca 2+ /CaM dependent phosphorylation of Histone II AS were determined as described. The data was analyzed using SIGMAPLOT and the fit to the data is represented. The data points are the mean of three measurements. Ó FEBS 2002 Chimeric calcium/calmodulin dependent protein kinase (Eur. J. Biochem. 269) 2459 0.998 (pH 8.5), 0.999 (pH 6.5), 0.992 (pH 7.5)]. The largest drop in enzyme activity occurred during the first 30 s of autophosphorylation (Fig. 2, Table 1). The enzyme activity continued to decrease, but the decrease was not as great as compared to the loss in the first 30 s. This suggests that there are two phases of inactivation. In the first phase (fast inactivation), the enzyme loses activity very rapidly and in the second phase (slow inactivation), loss of inactivation is significantly slower. Loss of enzyme activity showed these two phases of inactivation at all the pH conditions tried. 5 The autophosphorylation resulted in the formation of a sedimentable enzyme (Fig. 3). After 2 min of autophospho- rylation, CCaMK was detected in both the pellet and supernatant fractions. However, at time zero, all the kinase enzymes were seen in the supernatant, indicating that autophosphorylation leads to the formation of sedimentable CCaMK. The formation of a sedimentable enzyme was observed after 2 min of autophosphorylation at all the different reaction pH tested (Fig. 3). The role of the ATP concentration in sedimentablity of the enzyme was tested by conducting autophosphorylation at different ATP concen- trations (200 l M and 1 m M ). Figure 3 shows that a sedimentable enzyme was formed at pH 6.5 and 8.5 at both the ATP concentrations used for autophoshorylation. At pH 7.5, a higher ATP concentration (1 m M )prevented the formation of a sedimentable enzyme (Fig. 3). However, under all the other conditions of pH and ATP concentra- tions, kinase enzymes existed as both the sedimentable and soluble form. This suggests that both pH and ATP concentration determine sedimentability of CCaMK due to autophosphorylation. It is not necessary that all of the soluble enzyme remains unphosphorylated or all the phosphorylated enzyme undergoes sedimentation. This depends on pH and ATP concentration. At higher concen- trations of ATP (1 m M ) and at pH 7.5, all of the phosphorylated enzyme remains in the supernatant (soluble fraction). ATP concentration also influenced autophospho- rylation-dependent formation of sedimentable CaMK II [24]. Higher concentrations of ATP during autophospho- rylation prevented sedimentation of CaMK II. Inactivation was not accompanied by the formation of sedimentable CaMK II at different ATP concentrations at pH 7.5 [24]. In contrast, plant kinase showed the formation of a sediment- able enzyme at low concentrations of ATP (200 l M ), though higher concentrations inhibited formation of sedi- mentable enzyme as mentioned above. In conclusion, the inactivation of CCaMK depends on the duration of autophosphorylation, reaction pH and ATP concentration. The autophosphorylation-dependent inactivation was further studied by TEM. Figure 4A,B shows electron micrographs that are representative fields through out the entire grid and were observed only under the autophospho- rylation conditions that produced sedimentable enzyme. However, the percentage of sedimentable enzyme that adopted this network-like structure is uncertain. The autophosphorylated kinase after denaturation (by boiling in the presence of detergents) was observed under TEM. After denaturation, the autophosphorylated kinase did not show the formation of the network-like structures (data not shown) indicating that these complex structures are formed due to self-association caused by autophosphorylation. The role of ATP binding and ATP hydrolysis in the formation of the complexes was investigated by replacing the ATP with an unhydrolyzable ATP analogue, AMP- PNP, in the autophosphorylation reaction mix. Figure 4C shows that the network-like complexes are not formed in the presence of AMP-PNP. This suggests that ATP hydrolysis is required for the complex formation. The autophospho- rylation mutant T267A [12] did not show the complex formation (Fig. 4C) under similar conditions that produced complex structures of wild-type CCaMK. These results further suggest that the formation of the complexes seen under TEM is phosphorylation-dependent. Self-inactivation may be a mechanism of regulating enzyme activity as a means of modulating metabolic processes or signal transduction pathway. Self-inactivation imposes an upper limit on bioactive prostanoid synthesis by prostaglandin H synthase (PGHS) [25]. The cytochrome Fig. 3. Formation of sedimentable enzyme during autophosphorylation of CCaMK. CCaMK was autophosphorylated at different pH (6.5, 7.5 and 8.5) and at different ATP concentrations (200 l M and 1 m M )at0,2and 6 min. At the indicated time points, auto- phosphorylated enzyme was subjected to centrifugation and processing as described in Experimental procedures. The pellet (P) and supernatant (S) fractions are indicated at each time point. 2460 P. V. Sathyanarayanan and B. W. Poovaiah (Eur. J. Biochem. 269) Ó FEBS 2002 Fig. 4. Transmission electron micrographs of CCaMK after autophosphorylation showing the formation of complex structures. CCaMK was auto- phosphorylated for 5 min at pH 7.5 in the presence of 200 l M ATP and processed for TEM as described. (A) A field of uranyl acetate staining structures at X40 000 following autophosphorylation. (B) Complex structures due to autophosphorylation at ·100 000. (C) Autophosphorylation inthepresenceofAMP-PNPat·30 000. (D) Autophosphorylation of T267A, ·30 000. Arrows indicate polystyrene sizing beads (93 nm in diameter). Ó FEBS 2002 Chimeric calcium/calmodulin dependent protein kinase (Eur. J. Biochem. 269) 2461 P450 apoenzyme self-inactivation is accompanied by oxidation of all-groups, carbonyl group formation, changes in aggregate state and apoenzyme polymerization [26]. It is possible that heme loss and oxidative modification of the apoenzyme is an important step in the regulation of P450 decay in cell [26]. Redistribution of soluble CaM Kinase II to particulate fractions due to autophosphorylation has been demonstrated in both in vivo and in situ models of ischemia [27–29]. We have documented that the autophos- phorylation-dependent inactivation of CCaMK involves self-association leading to the formation of a sedimentable enzyme. Inactivation and subsequent formation of a sedimentable enzyme depends on the duration of auto- phosphorylation, reaction pH, and ATP concentration. To our knowledge, no other protein kinase reported from plants shows such a phosphorylation-dependent loss of activity. However, CCaMK-mediated protein phosphory- lation is implicated in male gametophyte development in plants [10]. Self-inactivation of CCaMK may be a mechan- ism of modulating the Ca 2+ /CaM mediated signal trans- duction pathway in anther. The elucidation of the molecular mechanisms leading to the self-inactivation of CCaMK will broaden our understanding of the regulation of Ca 2+ / CaM-mediated signaling in plants. ACKNOWLEDGEMENTS We thank Dr Chris Davitt and Professor Vincent Franceschi of the Electron Microscopy Center, WSU for their valuable help and suggestions with electron microscopy, and Shima Nakanishi for help with inactivation kinetics experiments. The support of the National Science Foundation (Grant MCB 0082256) and the National Aero- nautics and Space Administration (Grant NAG-10-0061) is gratefully acknowledged. REFERENCES 1. Poovaiah, B.W. & Reddy, A.S.N. (1993) Calcium and signal transduction in plants. CRC Crit. Rev. Plant. Sci. 12, 185–211. 2. Clapham, D.E. (1995) Calcium signalling. Cell. 80, 259–268. 3. Berridge, M.J., Lipp, P. & Bootman, M.D. (2000) The versatility and universality of calcium signaling. Nat Rev. Mol Cell Biol. 1, 11–21. 4. Roberts, D.M. & Harmon, A.C. (1992) Annu. Rev. 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Ó FEBS 2002 Chimeric calcium/calmodulin dependent protein kinase (Eur. J. Biochem. 269) 2463 . Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinase P. V. Sathyanarayanan and B. W study, we report autophosphorylation-dependent inactivation of CCaMK. Autophosphorylation of CCaMK resulted in time-dependent loss of enzyme activity (Figs