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BRSK2 is activated by cyclic AMP-dependent protein kinase A through phosphorylation at Thr260
BBRC Biochemical and Biophysical Research Communications 347 (2006) 867–871 www.elsevier.com/locate/ybbrc
BRSK2 is activated by cyclic AMP-dependent protein kinase A through phosphorylation at Thr260 Zekun Guo a
a,b
, Wenwen Tang a, Jian Yuan a, Xinya Chen a, Bo Wan a, Xiuting Gu a, Kuntian Luo a, Yingli Wang a, Long Yu a,*
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, PR China b School of Life Science, Northwest Sci-Tech University of Agriculture and Forestry, YangLing, Shaanxi, PR China Received 5 June 2006
Abstract Brain selective kinase 2 (BRSK2) has been identified as a member of AMPK related kinases. LKB1 can phosphorylate the Thr174 of BRSK2, increasing its activity >50-fold. In this study, we identified cAMP-dependent protein kinase A (PKA) as another upstream kinase of BRSK2, which can phosphorylate BRSK2 at Thr260. The association between these two proteins was confirmed by GST pull-down. Furthermore, our study indicated that the kinase activity of BRSK2 can be increased through phosphorylation by PKA. Ó 2006 Elsevier Inc. All rights reserved. Keywords: BRSK2; PKA; Phosphorylation; AMPK subfamily
BRSK2 (brain selective kinase 2) is a serine/threonine protein kinase and early study found it to be selectively expressed in brain. In 2002, BRSK2 was designated as a member of AMPK (AMP-activated protein kinase) subfamily of CAMK (Ca2+/calmodulin-dependent protein kinase) family and there are 14 members in this subfamily. Apart from BRSK2, other members are AMPKa1, AMPKa2, QSK, SIK, QIK, MARK1, MARK2, MARK3/Par-1A/CTAK1, MARK4, NUAK1/ARK5, NUAK2/SNARK, BRSK1/SAD-A, and MELK [1]. These members have been the new hotspot of research in recent years. The function of BRSK2 was previously inferred from the family members in model organisms. The orthologous gene in worm of BRSK2 is SAD-1, which has a role in presynapic vesicle clustering [2], suggesting that BRSK2 might have similar function. A highly conserved ascidian (chordate) homology of BRSK2 is also expressed in neural tissue and is asymmetrically localized to the posterior end of the embryo, suggesting that BRSK2 might play another role in *
Corresponding author. Fax: +86 21 65643250. E-mail address: [email protected] (L. Yu).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.178
embryonic axis determination [3]. Recently, KO mice for BRSK1 and BRSK2 (SAD kinases) have been reported [4]. Analysis of the mutant animals shows that these two kinases are required for forebrain neurons to acquire the polarity that endows axons and dendrites with distinct properties, which is promoted at least in part by phosphorylating the microtubule-associated protein Tau [4]. Subsequently, in 2006, BRSK1 and BRSK2 were also identified as novel SV- and active zone cytomatrix-associated protein kinases and reported to be involved in the regulation of neurotransmitter release, perhaps via phosphorylation of the active zone protein and vesicle priming factor RIM1 [5]. AMPK was first identified as physiological substrate of LKB1 [6–8] and LKB1 activates AMPK by phosphorylating Thr172 in the T-loop of this enzyme [9]. In 2004, Lizcano et al. reported that LKB1 can also phosphorylate the T-loop of other 11 members of AMPK subfamily, apart from MELK, increasing their activity >50-fold [10]. We report here that BRSK2 can be phosphorylated by PKA at Thr260 in vitro. Most interestingly, phosphorylation of BRSK2 by PKA activates its kinase activity. Thus,
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our findings uncover a novel mechanism of BRSK2 regulation. Materials and methods Plasmid construction and site-directed mutagenesis. All inserted fragments were obtained by PCR amplification and the oligonucleotide sequence of PCR primers is listed in Table 1. First, full-length BRSK2 was cloned into the eukaryotic expression vector pCMV-Myc at SfiI and XhoI sites using the primers Myc-BRSK2-F and Myc-BRSK2-R. It was also cloned into the prokaryotic expression vector pGEX-6P-1 at BamHI and SalI sites using the primers GST-BRSK2-F and GST-BRSK2-R. The kinase-dead mutation of BRSK2, in which Lys48 was replaced by Met, and other mutation of BRSK2 were constructed using the QuickChange site-directed mutagenesis kit (Stratagene, USA) following the manufacturer’s instructions. Cell culture, transfection, and lysis. HEK293T cells were cultured in a DMEM (Invitrogen) supplemented with 10% bovine serum (Invitrogen), 100 U/ml penicillin, and 100 lg/ml streptomycin. HEK293T cells were incubated in the environment of 37 °C in a humidified atmosphere containing 5% CO2. The cultured HEK293T cells were transfected with expression vectors using Lipofectamineä (Invitrogen) according to the protocol from Invitrogen. The cells were harvested at 48 h after transfection, washed twice with an ice-cold PBS, and then lysed on ice for 30 min in lysis buffer (Cell Signaling Technology) (supplemented with cocktail tablets and 0.1 mM PMSF) with gentle shaking. The solution was then centrifuged at 12,000g for 30 min at 4 °C to remove the debris, and supernatant was collected for use. Western blotting. Protein samples separated by SDS–PAGE were electro-transferred onto nitrocellulose membrane. The membrane was blocked at room temperature for 1 h with PBS containing 5% (w/v) skim milk and then washed with PBS containing 0.2% Tween 20 (Sigma; PBST). After blocking, the membrane was then incubated overnight at 4 °C with primary antibody diluted with PBS containing 1% (w/v) BSA. The membrane was washed with PBST and then incubated with HRP-conjugated secondary antibody at room temperature for 1 h. The membrane was washed again with PBST and then developed with ECL system (Santa Cruz). Expression and purification of BRSK2 from Escherichia coli. Unless otherwise indicated, pGEX constructs encoding full-length wild-type or mutant forms of GST-BRSK2 were transformed into E. coli BL21 cells. One-litre cultures were grown at 37 °C in Luria broth medium containing 100 lg/ml ampicillin until the absorbance at 600 nm reached 0.8. Two hundred micromolar isopropyl-b-D-galactoside was added, and the cells were cultured for a further 10 h at 25 °C. Pelleted cells were resuspended in 35 ml ice-cold PBS buffer containing 1 mg/ml lysozyme and lysed in one round of freeze/thaw, followed by sonication. The lysates were centrifuged for 30 min at 20,000g, and the GST fused BRSK2 was affinity purified on glutathione–Sepharose and on-column cleavage for 20 h at 4 °C with
ProScission Protease (Amersham Bioscience) to remove the GST tag. The target protein was then eluted with PBS buffer. The product was finally detected with 12% SDS–PAGE. Expression and purification of BRSK2 from HEK293T Cells. One 10 cm dish of HEK293T cells was cultured and transfected with 7 lg of the MycBRSK2 plasmid. Forty-eight hours after-transfection, the cells were lysed. Then the lysate was cultured with mouse anti-Myc monoclonal antibody and Protein G Plus/Protein A Agarose (Oncogene) and Myc-tagged BRSK2 was purified according to the standard immunoprecipitation protocol. GST-fusion protein pull-down experiments. The expression and purification of GST fusion proteins was done following the protocol of glutathione–Sepharose 4B (Amesham Phamacia Biotech). The purified protein GST or GST-PKA was covalently attached to the 50% slurry of glutathione–Sepharose beeds and then incubated with the whole-cell lysates of cells expression Myc-BRSK2 at 4 °C for 3 h in buffer containing 20 mM Tris–HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 0.5% NP-40, and protease inhibitors. The beeds were washed 5 times with washing buffer containing 20 mM Tris–HCl (pH 8.0), 500 mM NaCl, 1 mM EDTA, 1% NP-40, and protease inhibitors, and the bound proteins were analyzed by immunoblotting using mouse Myc-specific monoclonal antibody. SAMS phosphorylation assay. The prokaryotic expressed BRSK2 and Myc-tagged BRSK2 isolated from cell lysate were incubated, respectively, with kinase buffer (Cell Signaling Technology, USA), 2 lg substrate peptide (SAMS peptide: HMRSAMSGLHLVKRR or SAMA peptide: HMRSAMAGLHLVKRR), 10 lM ATP, and 1 ll [c-32P]-ATP (5 lCi). After incubation at 30 °C for 30 min, 15 ll aceticacidglacial was added to stop the reaction. Incorporation of 32P-phosphate into the peptide substrate was determined by applying the reaction mixture onto P81 phosphocellulose paper and scintillation counting after washing in phosphoric acid. In PKA phosphorylation assay, measured samples were separated on 12% SDA–PAGE for autoradiography.
Results BRSK2 was recently identified as a new serine/ threonine protein kinase, which belongs to the AMPK subfamily. In 2004, LKB1 was reported to phosphorylate and activate the 13 members of the AMPK subfamily, including BRSK2, in complex with the pseudokinase STRAD and the scaffolding protein MO25 [10]. Here, we analyzed the amino acid sequence of BRSK2 using Scansite (http://scansite.mit.edu) and found one potential site for PKA (illustrated in Fig. 1A and B). The potential PKA phosphorylation site, located at Thr260 of BRSK2, is conserved between the human and mouse Brsk2s (Fig. 1B).
Table 1 Oligonucleotides primers Primer
Sequeuce
Gene expression primer Myc-BRSK2-F Myc-BRSK2-R GST-BRSK2-F GST-BRSK2-R GST-PKA-F GST-PKA-R
5-ATGGCCATGGAGGCCATGACATCGACGGGGAAGG-3 5-ACACTCGAGTCAGAGGCTACTCTCGTAGCTGG-3 5-AGAGGATCCATGACATCGACGGGGAAGG-3 5-ACAGTCGACTCAGAGGCTACTCTCGTAGCTGG-3 5-ATAGGATCCATGGGGAACGCGGCGACCG-3 5-GCGGTCGACTTAAAATTCACCAAATTCTTTTGCAC-3
Mutation primer BRSK2-T260A-F BRSK2-T260A-R BRSK2-K48M-F BRSK2-K48M-R
5-CCGCACGCCGCCTCGCGCTAGAGCACATTC-3 5-GAATGTGCTCTAGCGCGAGGCGGCGTGCGG-3 5-GAAGGTGGCCATCATGATCGTCAACCGTGAG-3 5-CTCACGGTTGACGATCATGATGGCCACCTTC-3
The sequences underlined are the restriction sites and the mutation sites are shaded.
Z. Guo et al. / Biochemical and Biophysical Research Communications 347 (2006) 867–871
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A
B
C
Fig. 2. BRSK2 was phosphorylated by PKA at Thr260. (A) Kinase assay showing PKA phosphorylating recombinant BRSK2. The upper panel was the result of phosphorylation and the lower was SDS-PAGE stained with Coomassie brilliant blue R-250. (B) BRSK2 but not BRSK2-T260A was phosphorylated by PKA. The upper panel is X-ray film of kinase assays, and the lower panel is Coomassie brilliant blue R-250 staining (CBB) of the gel. Fig. 1. BRSK2 was predicted as a substrate of PKA. (A) Schematic of BRSK2 protein. The kinase domain of BRSK2 consists of amino acids 19–270, presented with black bold line. (B) Top, a potential PKA phosphorylation site in BRSK2 determined by database sequence analysis. Bottom, comparison of the human BRSK2 sequence with the mouse BRSK2 sequence surrounding Thr260. (C) Interaction of PKA and BRSK2 in vitro. The whole-cell lysates of cells expressing Myc-BRSK2 protein incubated with GST, or the GST-PKA fusion protein. After binding to glutathione–Sepharose beads, washing, and the final beadbound fractions were detected by Western blotting using Myc monoclonal antibody (upper panel). GST and GST-PKA proteins were detected by Western blotting using GST monoclonal antibody (lower panel).
In order to verify whether PKA can phosphorylate BRSK2 at Thr260, we first tested the ability of BRSK2 to interact with PKA using a GST pull-down experiment. No signal was observed after incubation of BRSK2 protein and GST protein but signal was observed after incubation of the BRSK2 protein with the GST-PKA fusion protein (Fig. 1C). The aforementioned experiment showed that BRSK2 can interact with PKA in vitro, and then we investigated whether PKA can phosphorylate BRSK2 in vitro using autoradiography. The recombinant catalytic subunit of PKA was from NEB and BRSK2 purified from bacteria was used as substrate. As shown in Fig. 2A, PKA can phosphorylate BRSK2 while the autophosphorylation of both PKA and BRSK2 was very weak. We then tried to identify whether the phosphorylation site on BRSK2 by PKA is Thr260 as predicted by software. We mutated the
Thr260 of BRSK2 into Ala and performed in vitro kinase assay using both BRSK2 wild-type and mutation as substrates. The band intensity of BRSK2 T260A mutation phosphorylated by PKA was very faint compared with that of BRSK2 wild-type (Fig. 2B), which suggested that PKA can phosphorylate BRSK2 at Thr260. LKB1 can phosphorylate BRSK2 at Thr174 and activate it [10]. Here our results showed that BRSK2 can also be phosphorylated by PKA at Thr260. Next, we further studied whether the phosphorylation of Thr260 by PKA would affect the kinase activity of BRSK2. The amino acid sequence of BRSK2’s kinase domain shares the highest similarity (65%) and identity (49%) with AMPK a1 subunit and a2 subunit in the kinome. Given that the substrate specificity of kinase is mainly dependent on amino acid sequence of its kinase domain [11,12], we used SAMS peptide, one of the substrates of AMPK, to analyze the kinase activity of BRSK2 with a control of SAMA peptide in which the Ser of SAMS peptide was mutated into Ala. Kinase assay showed that both BRSK2 purified from E. coli and that from HEK293T cells can phosphorylate SAMS peptide but not the control SAMA peptide (Fig. 3A) and the result is consistent with that from Lizcano et al. [10]. We then investigated the PKA’s effect on the kinase activity of BRSK2. BRSK2 was first phosphorylated by PKA, and the PKA was to be removed prior to kinase assay. For prokaryotic expressed BRSK2, 14–22 Amide (Cell-permeable, Myristoylated, PKA inhibitor, Calbiochem) was added into the reaction. And for
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Fig. 3. BRSK2 is activated by PKA through phosphorylation at Thr260. (A) Kinase assay of BRSK2 expressed in E. coli (Top) and Myc-tagged BRSK2 expressed in HEK293T cells (Middle). Bottom, The quantities of Myc-tagged proteins used in the kinase assay detected by western blot using c-Myc monoclonal antibody. (B) PKA phosphorylating Thr260 of BRSK2 expressed in E. coli (Top) or Myc-tagged BRSK2 expressed in HEK293T cells (Middle) induced BRSK2 activation. Recombinant proteins were first incubated with PKA and ATP in the reaction system for 1 h. 30 min before adding SAMS peptide, PKI was added into the reaction system (Top) or the immunoprecipitates were washed with kinase buffer three times (Middle). Then kinase assay was performed as previously described. Bottom, The quantities of Myc-tagged proteins used in the kinase assay detected by western blot using c-Myc monoclonal antibody. The kinase activity is the mean of four experiments and the bars represent SE value. Double asterisks indicate the statistical significance p < 0.01(t test).
Fig. 4. Alignment of the AMPK subfamily member sequences surrounding Thr174 (A) and Thr260 (B).
eukaryotic expressed BRSK2, the immunoprecipitates of BRSK2 were washed with kinase buffer for three times. Our results showed that PKA enhanced the kinase activity
of BRSK2 by about 2 times through phosphorylation at Thr260, whereas no visible effect was observed in the T260A mutation (Fig. 3B).
Z. Guo et al. / Biochemical and Biophysical Research Communications 347 (2006) 867–871
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Discussion
References
The AMP-activated protein kinase (AMPK) is an important regulator of cellular mechanism in response to metabolic stress and to other regulatory signals. The activation of AMPK not only needs allosteric regulation of AMP but also phosphorylation. Although that activation of AMPK requires phosphorylation was found in 1978, the molecular identification of AMPKK has remained elusive until recent advances made in the upstream kinases of SNF1, the homologue of AMPK in yeast [13,14]. A series of the following experiments proved that LKB1 is the upstream kinase which phosphorylates and activates AMPK at Thr172. Lizcano et al. [10] reported that LKB1 phosphorylates and activates the 13 members of the AMPK subfamily including BRSK2, in complex with the pseudokinase STRAD and the scaffolding protein MO25. Several lines of recent evidence point to the presence of non-LKB1 AMPKKs. Multiple AMPKK activities are separable during chromatography of extracts from rodent heart [15,16]. Since Manning et al. classified BRSK2 as a member of the AMPK subfamily in the CAMK family, the research on BRSK2 has been more and more. BRSK2 was not only found to be required for neuronal polarization [4] but also identified as a presynaptic kinase associated with synaptic vesicles and active zone cytomatrix that regulates neurotransmitter release [5]. Identification of the upstream kinases of BRSK2 can help further study the biological functions of BRSK2. Thr174 of BRSK2, which is phosphorylated by LKB1, is conserved during AMPK subfamily (Fig. 4A) and the phosphorylation at this site is necessary for the activity of the members of AMPK subfamily. Mutation of this site in these enzymes reduces the kinase activity [10,17,18]. In this study, we found a novel upstream kinase of BRSK2—PKA, which can phosphorylate Thr260 of BRSK2 in vitro and activate BRSK2. The Thr260 site phosphorylated by PKA is also highly conserved in the AMPK subfamily (Fig. 4B). This suggests a general regulatory mechanism exists in this site, similar to that of Thr174 in the AMPK subfamily. What is not known is whether PKA can really modulate the functions of BRSK2 in vivo. Also unclear is whether cross talk exists between the different phosphorylation sites of BRSK2, Thr174 by LKB1, and Thr260 by PKA. Further experiments are necessary to address these important issues.
[1] G. Manning, D.B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein kinase complement of the human genome, Science 298 (2002) 1912–1934. [2] J.G. Crump, M. Zhen, Y. Jin, C.I. Bargmann, The SAD-1 kinase regulates presynaptic vesicle clustering and axon termination, Neuron 29 (2001) 115–129. [3] Y. Sasakura, M. Ogasawara, K.W. Makabe, Maternally localized RNA encoding a serine/threonine protein kinase in the ascidian, Mech. Dev. 76 (1998) 161–163. [4] M. Kishi, Y.A. Pan, J.G. Crump, J.R. Sanes, Mammalian SAD kinases are required for neuronal polarization, Science 307 (2005) 929–932. [5] E. Inoue, S. Mochida, H. Takagi, S. Higa, M. Deguchi-Tawarada, E. Takao-Rikitsu, M. Inoue, I. Yao, K. Takeuchi, I. Kitajima, M. Setou, T. Ohtsuka, Y. Takai, SAD: a presynaptic kinase associated with synaptic vesicles and the active zone cytomatrix that regulates neurotransmitter release, Neuron 50 (2006) 261–275. [6] S.A. Hawley, J. Boudeau, J.L. Reid, K.J. Mustard, L. Udd, T.P. Makela, D.R. Alessi, D.G. Hardie, Complexes between the LKB1 tumor suppressor, STRADalpha/beta and MO25alpha/beta are upstream kinases in the AMP-activated protein kinase cascade, J. Biol. 2 (2003) 28. [7] R.J. Shaw, M. Kosmatka, N. Bardeesy, R.L. Hurley, L.A. Witters, R.A. DePinho, L.C. Cantley, The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress, Proc. Natl. Acad. Sci. USA 101 (2004) 3329–3335. [8] A. Woods, S.R. Johnstone, K. Dickerson, F.C. Leiper, L.G. Fryer, D. Neumann, U. Schlattner, T. Wallimann, M. Carlson, D. Carling, LKB1 is the upstream kinase in the AMP-activated protein kinase cascade, Curr. Biol. 13 (2003) 2004–2008. [9] D.G. Hardie, The AMP-activated protein kinase pathway—newplayers upstream and downstream, J. Cell Sci. 117 (2004) 5479–5487. [10] J.M. Lizcano, O. Goransson, R. Toth, M. Deak, N.A. Morrice, J. Boudeau, S.A. Hawley, L. Udd, T.P. Makela, D.G. Hardie, D.R. Alessi, LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1, EMBO J. 23 (2004) 833–843. [11] R.I. Brinkworth, R.A. Breinl, B. Kobe, Structural basis and prediction of substrate specificity in protein serine/threonine kinases, Proc. Natl. Acad. Sci. USA 100 (2003) 74–79. [12] L. Li, E.I. Shakhnovich, L.A. Mirny, Amino acids determining enzyme-substrate specificity in prokaryotic and eukaryotic protein kinases, Proc. Natl. Acad. Sci. USA 100 (2003) 4463–4468. [13] S.P. Hong, F.C. Leiper, A. Woods, D. Carling, M. Carlson, Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases, Proc. Natl. Acad. Sci. USA 100 (2003) 8839–8843. [14] C.M. Sutherland, S.A. Hawley, R.R. McCartney, A. Leech, M.J. Stark, M.C. Schmidt, D.G. Hardie, Elm1p is one of three upstream kinases for the Saccharomyces cerevisiae SNF1 complex, Curr. Biol. 13 (2003) 1299–1305. [15] J.Y. Altarejos, M. Taniguchi, A.S. Clanachan, G.D. Lopaschuk, Myocardial ischemia differentially regulates LKB1 and an alternate 5 0 -AMP-activated protein kinase, J. Biol. Chem. 280 (2005) 183–190. [16] S.J. Baron, J. Li, R.R. Russell, D. Neumann, E.J. Miller, R. Tuerk, T. Wallimann, R.L. Hurley, L.A. Witters, L.H. Young, Dual mechanisms regulating AMPK kinase action in the ischemic heart, Circ. Res. 96 (2005) 337–345. [17] S.C. Stein, A. Woods, N.A. Jones, M.D. Davison, D. Carling, The regulation of AMP-activated protein kinase phosphorylation, Biochem. J. 345 (2000) 437–443. [18] B.E. Crute, K. Seefeld, J. Gamble, B.E. Kemp, L.A. Witters, Functional domains of the a1 catalytic subunit of the AMP-activated protein kinase, J. Biol. Chem. 273 (1998) 35347–35354.
Acknowledgments This work was supported by the National 973 program of China, 863 projects of China, and the National Natural Science foundation of China (30024001).
BRSK2 is activated by cyclic AMP-dependent protein kinase A through phosphorylation at Thr260 Zekun Guo a
a,b
, Wenwen Tang a, Jian Yuan a, Xinya Chen a, Bo Wan a, Xiuting Gu a, Kuntian Luo a, Yingli Wang a, Long Yu a,*
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, PR China b School of Life Science, Northwest Sci-Tech University of Agriculture and Forestry, YangLing, Shaanxi, PR China Received 5 June 2006
Abstract Brain selective kinase 2 (BRSK2) has been identified as a member of AMPK related kinases. LKB1 can phosphorylate the Thr174 of BRSK2, increasing its activity >50-fold. In this study, we identified cAMP-dependent protein kinase A (PKA) as another upstream kinase of BRSK2, which can phosphorylate BRSK2 at Thr260. The association between these two proteins was confirmed by GST pull-down. Furthermore, our study indicated that the kinase activity of BRSK2 can be increased through phosphorylation by PKA. Ó 2006 Elsevier Inc. All rights reserved. Keywords: BRSK2; PKA; Phosphorylation; AMPK subfamily
BRSK2 (brain selective kinase 2) is a serine/threonine protein kinase and early study found it to be selectively expressed in brain. In 2002, BRSK2 was designated as a member of AMPK (AMP-activated protein kinase) subfamily of CAMK (Ca2+/calmodulin-dependent protein kinase) family and there are 14 members in this subfamily. Apart from BRSK2, other members are AMPKa1, AMPKa2, QSK, SIK, QIK, MARK1, MARK2, MARK3/Par-1A/CTAK1, MARK4, NUAK1/ARK5, NUAK2/SNARK, BRSK1/SAD-A, and MELK [1]. These members have been the new hotspot of research in recent years. The function of BRSK2 was previously inferred from the family members in model organisms. The orthologous gene in worm of BRSK2 is SAD-1, which has a role in presynapic vesicle clustering [2], suggesting that BRSK2 might have similar function. A highly conserved ascidian (chordate) homology of BRSK2 is also expressed in neural tissue and is asymmetrically localized to the posterior end of the embryo, suggesting that BRSK2 might play another role in *
Corresponding author. Fax: +86 21 65643250. E-mail address: [email protected] (L. Yu).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.178
embryonic axis determination [3]. Recently, KO mice for BRSK1 and BRSK2 (SAD kinases) have been reported [4]. Analysis of the mutant animals shows that these two kinases are required for forebrain neurons to acquire the polarity that endows axons and dendrites with distinct properties, which is promoted at least in part by phosphorylating the microtubule-associated protein Tau [4]. Subsequently, in 2006, BRSK1 and BRSK2 were also identified as novel SV- and active zone cytomatrix-associated protein kinases and reported to be involved in the regulation of neurotransmitter release, perhaps via phosphorylation of the active zone protein and vesicle priming factor RIM1 [5]. AMPK was first identified as physiological substrate of LKB1 [6–8] and LKB1 activates AMPK by phosphorylating Thr172 in the T-loop of this enzyme [9]. In 2004, Lizcano et al. reported that LKB1 can also phosphorylate the T-loop of other 11 members of AMPK subfamily, apart from MELK, increasing their activity >50-fold [10]. We report here that BRSK2 can be phosphorylated by PKA at Thr260 in vitro. Most interestingly, phosphorylation of BRSK2 by PKA activates its kinase activity. Thus,
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our findings uncover a novel mechanism of BRSK2 regulation. Materials and methods Plasmid construction and site-directed mutagenesis. All inserted fragments were obtained by PCR amplification and the oligonucleotide sequence of PCR primers is listed in Table 1. First, full-length BRSK2 was cloned into the eukaryotic expression vector pCMV-Myc at SfiI and XhoI sites using the primers Myc-BRSK2-F and Myc-BRSK2-R. It was also cloned into the prokaryotic expression vector pGEX-6P-1 at BamHI and SalI sites using the primers GST-BRSK2-F and GST-BRSK2-R. The kinase-dead mutation of BRSK2, in which Lys48 was replaced by Met, and other mutation of BRSK2 were constructed using the QuickChange site-directed mutagenesis kit (Stratagene, USA) following the manufacturer’s instructions. Cell culture, transfection, and lysis. HEK293T cells were cultured in a DMEM (Invitrogen) supplemented with 10% bovine serum (Invitrogen), 100 U/ml penicillin, and 100 lg/ml streptomycin. HEK293T cells were incubated in the environment of 37 °C in a humidified atmosphere containing 5% CO2. The cultured HEK293T cells were transfected with expression vectors using Lipofectamineä (Invitrogen) according to the protocol from Invitrogen. The cells were harvested at 48 h after transfection, washed twice with an ice-cold PBS, and then lysed on ice for 30 min in lysis buffer (Cell Signaling Technology) (supplemented with cocktail tablets and 0.1 mM PMSF) with gentle shaking. The solution was then centrifuged at 12,000g for 30 min at 4 °C to remove the debris, and supernatant was collected for use. Western blotting. Protein samples separated by SDS–PAGE were electro-transferred onto nitrocellulose membrane. The membrane was blocked at room temperature for 1 h with PBS containing 5% (w/v) skim milk and then washed with PBS containing 0.2% Tween 20 (Sigma; PBST). After blocking, the membrane was then incubated overnight at 4 °C with primary antibody diluted with PBS containing 1% (w/v) BSA. The membrane was washed with PBST and then incubated with HRP-conjugated secondary antibody at room temperature for 1 h. The membrane was washed again with PBST and then developed with ECL system (Santa Cruz). Expression and purification of BRSK2 from Escherichia coli. Unless otherwise indicated, pGEX constructs encoding full-length wild-type or mutant forms of GST-BRSK2 were transformed into E. coli BL21 cells. One-litre cultures were grown at 37 °C in Luria broth medium containing 100 lg/ml ampicillin until the absorbance at 600 nm reached 0.8. Two hundred micromolar isopropyl-b-D-galactoside was added, and the cells were cultured for a further 10 h at 25 °C. Pelleted cells were resuspended in 35 ml ice-cold PBS buffer containing 1 mg/ml lysozyme and lysed in one round of freeze/thaw, followed by sonication. The lysates were centrifuged for 30 min at 20,000g, and the GST fused BRSK2 was affinity purified on glutathione–Sepharose and on-column cleavage for 20 h at 4 °C with
ProScission Protease (Amersham Bioscience) to remove the GST tag. The target protein was then eluted with PBS buffer. The product was finally detected with 12% SDS–PAGE. Expression and purification of BRSK2 from HEK293T Cells. One 10 cm dish of HEK293T cells was cultured and transfected with 7 lg of the MycBRSK2 plasmid. Forty-eight hours after-transfection, the cells were lysed. Then the lysate was cultured with mouse anti-Myc monoclonal antibody and Protein G Plus/Protein A Agarose (Oncogene) and Myc-tagged BRSK2 was purified according to the standard immunoprecipitation protocol. GST-fusion protein pull-down experiments. The expression and purification of GST fusion proteins was done following the protocol of glutathione–Sepharose 4B (Amesham Phamacia Biotech). The purified protein GST or GST-PKA was covalently attached to the 50% slurry of glutathione–Sepharose beeds and then incubated with the whole-cell lysates of cells expression Myc-BRSK2 at 4 °C for 3 h in buffer containing 20 mM Tris–HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 0.5% NP-40, and protease inhibitors. The beeds were washed 5 times with washing buffer containing 20 mM Tris–HCl (pH 8.0), 500 mM NaCl, 1 mM EDTA, 1% NP-40, and protease inhibitors, and the bound proteins were analyzed by immunoblotting using mouse Myc-specific monoclonal antibody. SAMS phosphorylation assay. The prokaryotic expressed BRSK2 and Myc-tagged BRSK2 isolated from cell lysate were incubated, respectively, with kinase buffer (Cell Signaling Technology, USA), 2 lg substrate peptide (SAMS peptide: HMRSAMSGLHLVKRR or SAMA peptide: HMRSAMAGLHLVKRR), 10 lM ATP, and 1 ll [c-32P]-ATP (5 lCi). After incubation at 30 °C for 30 min, 15 ll aceticacidglacial was added to stop the reaction. Incorporation of 32P-phosphate into the peptide substrate was determined by applying the reaction mixture onto P81 phosphocellulose paper and scintillation counting after washing in phosphoric acid. In PKA phosphorylation assay, measured samples were separated on 12% SDA–PAGE for autoradiography.
Results BRSK2 was recently identified as a new serine/ threonine protein kinase, which belongs to the AMPK subfamily. In 2004, LKB1 was reported to phosphorylate and activate the 13 members of the AMPK subfamily, including BRSK2, in complex with the pseudokinase STRAD and the scaffolding protein MO25 [10]. Here, we analyzed the amino acid sequence of BRSK2 using Scansite (http://scansite.mit.edu) and found one potential site for PKA (illustrated in Fig. 1A and B). The potential PKA phosphorylation site, located at Thr260 of BRSK2, is conserved between the human and mouse Brsk2s (Fig. 1B).
Table 1 Oligonucleotides primers Primer
Sequeuce
Gene expression primer Myc-BRSK2-F Myc-BRSK2-R GST-BRSK2-F GST-BRSK2-R GST-PKA-F GST-PKA-R
5-ATGGCCATGGAGGCCATGACATCGACGGGGAAGG-3 5-ACACTCGAGTCAGAGGCTACTCTCGTAGCTGG-3 5-AGAGGATCCATGACATCGACGGGGAAGG-3 5-ACAGTCGACTCAGAGGCTACTCTCGTAGCTGG-3 5-ATAGGATCCATGGGGAACGCGGCGACCG-3 5-GCGGTCGACTTAAAATTCACCAAATTCTTTTGCAC-3
Mutation primer BRSK2-T260A-F BRSK2-T260A-R BRSK2-K48M-F BRSK2-K48M-R
5-CCGCACGCCGCCTCGCGCTAGAGCACATTC-3 5-GAATGTGCTCTAGCGCGAGGCGGCGTGCGG-3 5-GAAGGTGGCCATCATGATCGTCAACCGTGAG-3 5-CTCACGGTTGACGATCATGATGGCCACCTTC-3
The sequences underlined are the restriction sites and the mutation sites are shaded.
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A
B
C
Fig. 2. BRSK2 was phosphorylated by PKA at Thr260. (A) Kinase assay showing PKA phosphorylating recombinant BRSK2. The upper panel was the result of phosphorylation and the lower was SDS-PAGE stained with Coomassie brilliant blue R-250. (B) BRSK2 but not BRSK2-T260A was phosphorylated by PKA. The upper panel is X-ray film of kinase assays, and the lower panel is Coomassie brilliant blue R-250 staining (CBB) of the gel. Fig. 1. BRSK2 was predicted as a substrate of PKA. (A) Schematic of BRSK2 protein. The kinase domain of BRSK2 consists of amino acids 19–270, presented with black bold line. (B) Top, a potential PKA phosphorylation site in BRSK2 determined by database sequence analysis. Bottom, comparison of the human BRSK2 sequence with the mouse BRSK2 sequence surrounding Thr260. (C) Interaction of PKA and BRSK2 in vitro. The whole-cell lysates of cells expressing Myc-BRSK2 protein incubated with GST, or the GST-PKA fusion protein. After binding to glutathione–Sepharose beads, washing, and the final beadbound fractions were detected by Western blotting using Myc monoclonal antibody (upper panel). GST and GST-PKA proteins were detected by Western blotting using GST monoclonal antibody (lower panel).
In order to verify whether PKA can phosphorylate BRSK2 at Thr260, we first tested the ability of BRSK2 to interact with PKA using a GST pull-down experiment. No signal was observed after incubation of BRSK2 protein and GST protein but signal was observed after incubation of the BRSK2 protein with the GST-PKA fusion protein (Fig. 1C). The aforementioned experiment showed that BRSK2 can interact with PKA in vitro, and then we investigated whether PKA can phosphorylate BRSK2 in vitro using autoradiography. The recombinant catalytic subunit of PKA was from NEB and BRSK2 purified from bacteria was used as substrate. As shown in Fig. 2A, PKA can phosphorylate BRSK2 while the autophosphorylation of both PKA and BRSK2 was very weak. We then tried to identify whether the phosphorylation site on BRSK2 by PKA is Thr260 as predicted by software. We mutated the
Thr260 of BRSK2 into Ala and performed in vitro kinase assay using both BRSK2 wild-type and mutation as substrates. The band intensity of BRSK2 T260A mutation phosphorylated by PKA was very faint compared with that of BRSK2 wild-type (Fig. 2B), which suggested that PKA can phosphorylate BRSK2 at Thr260. LKB1 can phosphorylate BRSK2 at Thr174 and activate it [10]. Here our results showed that BRSK2 can also be phosphorylated by PKA at Thr260. Next, we further studied whether the phosphorylation of Thr260 by PKA would affect the kinase activity of BRSK2. The amino acid sequence of BRSK2’s kinase domain shares the highest similarity (65%) and identity (49%) with AMPK a1 subunit and a2 subunit in the kinome. Given that the substrate specificity of kinase is mainly dependent on amino acid sequence of its kinase domain [11,12], we used SAMS peptide, one of the substrates of AMPK, to analyze the kinase activity of BRSK2 with a control of SAMA peptide in which the Ser of SAMS peptide was mutated into Ala. Kinase assay showed that both BRSK2 purified from E. coli and that from HEK293T cells can phosphorylate SAMS peptide but not the control SAMA peptide (Fig. 3A) and the result is consistent with that from Lizcano et al. [10]. We then investigated the PKA’s effect on the kinase activity of BRSK2. BRSK2 was first phosphorylated by PKA, and the PKA was to be removed prior to kinase assay. For prokaryotic expressed BRSK2, 14–22 Amide (Cell-permeable, Myristoylated, PKA inhibitor, Calbiochem) was added into the reaction. And for
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Fig. 3. BRSK2 is activated by PKA through phosphorylation at Thr260. (A) Kinase assay of BRSK2 expressed in E. coli (Top) and Myc-tagged BRSK2 expressed in HEK293T cells (Middle). Bottom, The quantities of Myc-tagged proteins used in the kinase assay detected by western blot using c-Myc monoclonal antibody. (B) PKA phosphorylating Thr260 of BRSK2 expressed in E. coli (Top) or Myc-tagged BRSK2 expressed in HEK293T cells (Middle) induced BRSK2 activation. Recombinant proteins were first incubated with PKA and ATP in the reaction system for 1 h. 30 min before adding SAMS peptide, PKI was added into the reaction system (Top) or the immunoprecipitates were washed with kinase buffer three times (Middle). Then kinase assay was performed as previously described. Bottom, The quantities of Myc-tagged proteins used in the kinase assay detected by western blot using c-Myc monoclonal antibody. The kinase activity is the mean of four experiments and the bars represent SE value. Double asterisks indicate the statistical significance p < 0.01(t test).
Fig. 4. Alignment of the AMPK subfamily member sequences surrounding Thr174 (A) and Thr260 (B).
eukaryotic expressed BRSK2, the immunoprecipitates of BRSK2 were washed with kinase buffer for three times. Our results showed that PKA enhanced the kinase activity
of BRSK2 by about 2 times through phosphorylation at Thr260, whereas no visible effect was observed in the T260A mutation (Fig. 3B).
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Discussion
References
The AMP-activated protein kinase (AMPK) is an important regulator of cellular mechanism in response to metabolic stress and to other regulatory signals. The activation of AMPK not only needs allosteric regulation of AMP but also phosphorylation. Although that activation of AMPK requires phosphorylation was found in 1978, the molecular identification of AMPKK has remained elusive until recent advances made in the upstream kinases of SNF1, the homologue of AMPK in yeast [13,14]. A series of the following experiments proved that LKB1 is the upstream kinase which phosphorylates and activates AMPK at Thr172. Lizcano et al. [10] reported that LKB1 phosphorylates and activates the 13 members of the AMPK subfamily including BRSK2, in complex with the pseudokinase STRAD and the scaffolding protein MO25. Several lines of recent evidence point to the presence of non-LKB1 AMPKKs. Multiple AMPKK activities are separable during chromatography of extracts from rodent heart [15,16]. Since Manning et al. classified BRSK2 as a member of the AMPK subfamily in the CAMK family, the research on BRSK2 has been more and more. BRSK2 was not only found to be required for neuronal polarization [4] but also identified as a presynaptic kinase associated with synaptic vesicles and active zone cytomatrix that regulates neurotransmitter release [5]. Identification of the upstream kinases of BRSK2 can help further study the biological functions of BRSK2. Thr174 of BRSK2, which is phosphorylated by LKB1, is conserved during AMPK subfamily (Fig. 4A) and the phosphorylation at this site is necessary for the activity of the members of AMPK subfamily. Mutation of this site in these enzymes reduces the kinase activity [10,17,18]. In this study, we found a novel upstream kinase of BRSK2—PKA, which can phosphorylate Thr260 of BRSK2 in vitro and activate BRSK2. The Thr260 site phosphorylated by PKA is also highly conserved in the AMPK subfamily (Fig. 4B). This suggests a general regulatory mechanism exists in this site, similar to that of Thr174 in the AMPK subfamily. What is not known is whether PKA can really modulate the functions of BRSK2 in vivo. Also unclear is whether cross talk exists between the different phosphorylation sites of BRSK2, Thr174 by LKB1, and Thr260 by PKA. Further experiments are necessary to address these important issues.
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Acknowledgments This work was supported by the National 973 program of China, 863 projects of China, and the National Natural Science foundation of China (30024001).