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Genes to Cells (2004) 9, 219-232. doi:10.1111/j.1356-9597.2004.00714.x
© 2004 Blackwell Publishing or its licensors
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Involvement of BREK, a serine/threonine kinase enriched in brain, in NGF signalling

Seiji Kawa1, Jiro Fujimoto1, Tohru Tezuka1, Takanobu Nakazawa1 and Tadashi Yamamoto1,*

1 Department of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
We identified AATYK2 (Apoptosis-Associated Tyrosine Kinase 2) through a database search as a kinase specifically expressed in the brain. After characterization, we renamed it BREK (Brain-Enriched Kinase). Mouse BREK mRNA is expressed predominantly in brain, especially in olfactory bulb, olfactory tubercle, hippocampus, striatum, cerebellum, and cerebral cortex. Levels of expression and phosphorylation of BREK were high at 0–2 weeks after birth, suggesting that BREK is involved in neural development and functions during the early postnatal period. Phosphoamino acid analysis following in vitro kinase reaction revealed that BREK is a catalytically active, serine/threonine kinase. In PC12 cells, BREK was phosphorylated rapidly upon stimulation with nerve growth factor (NGF) in a protein kinase C-dependent pathway. In differentiated PC12 cells, BREK was enriched in cell bodies and growth cones, and also present along neurites. Introduction of a kinase-defective mutant of BREK into PC12 cells enhanced both ERK phosphorylation and neurite outgrowth in response to NGF, suggesting that BREK is a negative regulator of NGF-induced neuronal differentiation. Thus, we conclude that BREK is a new member of the family of protein serine/threonine kinases and that it plays important roles in NGF-TrkA signalling.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Protein-phosphorylation reactions play pivotal roles in regulating intracellular signals in response to a wide variety of environmental changes. There is accumulating evidence that protein kinases have critical and distinct roles in neural function. For example, Ca2+/calmodulin-dependent protein kinase II (CaMK II) is a major mediator of calcium signalling required for a variety of events in neurones. The events include neurotransmitter synthesis and release, modulation of neurotransmitter receptors and ion channels, and expression of genes associated with several aspects of synaptic plasticity, including long-term potentiation and spatial learning (Hanson & Schulman 1992; Miyamoto & Fukunaga 1996; Soderling 1996; Tan & Liang 1996). TrkA, which is a high-affinity neurotrophin receptor tyrosine kinase, also has critical roles in neuronal cells. TrkA mediates the actions of nerve growth factor (NGF) on subsets of sympathetic and sensory neurones as well as forebrain cholinergic neurones (Levi-Montalcini 1987; Lewin & Barde 1996; Thoenen 1995; Bonhoeffer 1996). Mice lacking TrkA showed loss of paravertebral sympathetic neurones (Snider 1994), suggesting the importance of NGF/TrkA signalling in neuronal development.

Although the sequencing of the human genome has revealed virtually all of the protein kinases encoded therein, the functions of many of these kinases expressed in the nervous system are presently unknown. Identification and functional analysis of kinases expressed in brain will provide the new insights into the molecular mechanisms of neural function. Here, we describe identification of a kinase, termed BREK (Brain-Enriched Kinase), through a database search. The kinase domain of BREK showed highest homology to that of AATYK1 (Apoptosis-Associated Tyrosine Kinase 1), which is reported to be a tyrosine kinase induced by apoptosis of myeloid cells (Gazzoa et al. 1997). In the list of human tyrosine kinases, there are genes encoding other kinases tentatively termed AATYK2 and AATYK3 that are homologous to AATYK1 and thought to be tyrosine kinases (Robinson et al. 2000). During the course of our study, a kinase termed KPI-2 (Kinase/Phosphatase/Inhibitor-2) or CPRK (CDK5/p35-Regulated Kinase), which was turned out to be identical to AATYK2, was reported to interact with PP1C and p35 (Wang & Brautigan 2002; Kesavapany et al. 2003). However, their in vivo associations and biological significances are little characterized. Here we report that BREK is the same protein as AATYK2/KPI-2/CPRK and is expressed primarily in early postnatal brain and, as well as AATYK1 and 3, has a serine/threonine kinase activity. We further show that BREK plays a part in NGF signalling in PC12 cells. Our findings suggest that BREK is involved in neural development and function in early postnatal development.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Identification and sequence analysis of BREK

In the course of a database search for novel kinases expressed in brain, we found that KIAA1079 (GENBANK Accession No. AB029002), which is contained in the HUGE database (Kazusa DNA Research Institute), encodes a putative novel kinase. The protein product of is identical to that of , which is listed in the catalogue of human protein tyrosine kinases (Robinson et al. 2000). We also found that encodes , listed in the catalogue (Robinson et al. 2000). RT-PCR data in this database showed that the mRNA is expressed mainly in brain. We confirmed by Northern blot analysis that the mouse homolog of is expressed predominantly in brain (see below). In addition, we showed that encodes a serine/threonine kinase (see below). Therefore, we renamed the protein encoded by Brain-Enriched Kinase (BREK).

Human BREK comprises 1503 amino acids (Wang & Brautigan 2002). The primary structure of BREK is shown schematically in Fig. 1A. The N-terminal region (residues 137–407) of human BREK has strong homology to tyrosine kinases (Hanks & Hunter 1995). Computer analysis revealed that this sequence contains a DLALRN-motif (residues 265–270), which is conserved in subdomain VI of non-Src tyrosine kinases. Although an arginine residue is usually located just N-terminal to the DLALRN motif in tyrosine kinases, a serine residue is present at this position in BREK (Ser264) as well as in several serine/threonine kinases. The phenylalanine in the DFG motif, which is located in subdomain VII of tyrosine kinases, is changed to a tyrosine (Tyr284) in BREK. We also found a putative autophosphorylation site at residue 295 that corresponds to a conserved tyrosine autophosphorylation site in Src-family tyrosine kinases. These characteristic amino acids are also present in AATYK1 and AATYK3 (Fig. 1B). The C-terminal region of BREK was rich in proline residues and contained seven PXXP-motifs that are potential SH3 domain binding sites. In addition, computer analysis revealed that there are two hydrophobic sequences, residues 11–32 and 46–68, in tandem at the N-terminus. A region just N-terminal of the hydrophobic sequence (11–32) is rich in basic amino acids (Arg8Arg9Arg10), suggesting this sequence may serve as a signal peptide. These characteristics, two hydrophobic sequences preceded by a basic amino acid-rich sequence, are also found in AATYK3 (Fig. 1A) but are not present in the corresponding region of AATYK1. AATYK1 is reported to be a cytosolic protein (Raghunath et al. 2000). These sequences may regulate subcellular localization (facilitate membrane localization) of BREK and AATYK3.



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  		Figure 1  Sequence analysis of human BREK. (A) schematic representation of putative protein structures of AATYK family kinases. The percentages of amino acid identities in the kinase domains are indicated. Kinase domains, hydrophobic regions, and PXXP motifs are shown by dotted boxes, hatched boxes, and solid lines, respectively. (B) sequence alignments of the kinase domains of human BREK, TrkA, and c-Src (left), and those of BREK, AATYK1, and AATYK3 (right). Conserved motifs are shown by the boxes with solid lines. Aspartic acid at residue 265 is shown in bold, which was mutated to valine to make a kinase-defective mutant.

 
Characterization of anti-BREK antibodies

We generated rabbit polyclonal antibodies, ma2 and ma4, against mouse BREK and 217C against human BREK. Full-length human BREK was expressed as a FLAG-tagged protein in 293T cells, and the cell lysates were subjected to immunoblotting. Both 217C and anti-FLAG antibody recognized a protein migrating at approximately 300 kD on SDS-PAGE gels (Fig. 2A,B). Both anti-mouse BREK antibodies, ma2 and ma4, also recognized a ~300 kD protein that was expressed in adult and embryonic mouse brain but not in adult mouse liver (Fig. 2C,D). Note that expression of BREK mRNA is prominent in brain and very low in liver (see below). Signals were absent when the antibodies were preabsorbed with appropriate antigens (Fig. 2C,D). In addition, the ~300 kD protein immunoprecipitated from mouse brain lysates with ma4 was also detected by ma2 (Fig. 2E). Taken together, these data suggested that the ~300 kD protein recognized by ma2, ma4, and 217C is the protein product of BREK. Because the calculated molecular weight of human BREK is 165 k, BREK might undergo some post-translational modifications in addition to phosphorylation. Alternatively, low electrophoretic mobility of BREK could be attributed to its C-terminal proline-rich region, since proline-rich proteins often migrate slower than expected on SDS-PAGE. The amino acid sequences of the BREK antigens were not conserved between human and mouse. Accordingly, 217C did not recognize rodent BREK, and ma2 and ma4 did not recognize human BREK (data not shown).



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  		Figure 2  Characterization of the antibodies against BREK. (A, B) Lysates of 293T cells expressing FLAG-tagged human BREK were subjected to Western blot analysis using the anti-human BREK antibodies, 217C (A) or the anti-FLAG antibody (B). (C, D) Western blot analysis of mouse tissue lysates using the anti-mouse BREK antibodies, ma2 (C) or ma4 (D). Antibodies (1 µg) were preincubated with 5 µg of GST (–) or the antigen fused to GST (+), and then used for Western blotting. (E) Mouse brain lysates were immunoprecipitated with ma2 or ma4. Immunoprecipitates were subjected to Western blot analysis using ma2.

 
Temporal and spatial expression of mouse BREK

We performed Northern blot analysis of total RNAs from various mouse tissues using mouse BREK cDNA as a probe (Fig. 3A). We detected a single transcript in all tested tissues, with the most prominent signal being in telencephalon. Other reports showed strong expression of human KPI-2/BREK mRNA in skeletal muscle rather than in brain (Wang & Brautigan 2002). However, by RT-PCR, we confirmed that the expression of mouse BREK mRNA is much higher in brain than in skeletal muscle. The PCR-amplified region corresponded to a portion of mouse BREK cDNA that is different from that used as the probe for Northern blot analysis. Thus, BREK is predominantly expressed in brain, at least, among mouse tissues.



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  		Figure 3  mRNA and protein expression of mouse BREK. (A) tissue specific expression of BREK mRNA. Total RNAs from adult mouse tissues were hybridized with a mouse BREK cDNA probe. The quality and quantity of RNAs were verified by ethidium bromide staining. (B) the temporal expression pattern of mouse BREK protein in brain. Protein lysates from mouse brains at indicated ages were separated by 5.5% SDS-PAGE and analysed by Western blotting using anti-mouse BREK antibodies, ma2 (top). Similar results were obtained using ma4. The amounts of lysates were verified by anti-{alpha}-tubulin blotting (bottom). (C) phosphorylation-dependent mobility shift of BREK protein during development. Mouse brain lysates at indicated ages were mock-treated, or treated with CIAP, and then subjected to Western blotting using ma2. Similar results were obtained by using ma4.

 
To evaluate the temporal expression of BREK in brain, we performed Western blot analysis of lysates of mouse brain at various developmental stages using antibodies against mouse BREK (Fig. 3B). BREK expression was observed during all tested stages from embryonic day 18 (E18) to postnatal week 6, but it was especially high during the early postnatal stage (postnatal weeks 0–2). In addition, we found that the mobility of BREK protein on SDS-PAGE gels was lower during early development (E18 to postnatal week 2, see also Fig. 2). The low mobility of BREK at E18 was altered by treatment with calf intestinal alkaline phosphatase (CIAP) (Fig. 3C), suggesting that the mobility shift was due to phosphorylation. Both the level of expression and the phosphorylation status of BREK suggested that it functions in the early postnatal brain. To determine the precise expression pattern of BREK mRNA in brain, we performed in situ hybridization analysis of adult mouse brain sections (postnatal week 6). AATYK1 and AATYK3 mRNAs were also examined (Fig. 4A). Expression of both BREK and AATYK3 was prominent in olfactory bulb, olfactory tubercle, hippocampus, striatum, cerebellum, and cerebral cortex. In contrast, AATYK1 was expressed ubiquitously in all regions of brain as reported previously (Tomomura et al. 2001). Although both BREK and AATYK3 were expressed mainly in the neuronal layers of forebrain, they showed distinct cellular patterns of expression (Fig. 4B). In olfactory bulb, BREK was expressed exclusively in mitral cells and glomeruli, whereas AATYK3 was expressed in granule cells as well as in mitral cells and glomeruli. In hippocampus, AATYK3 was expressed in both the CA1-CA3 region and dentate gyrus at similar levels, whereas expression of BREK in dentate gyrus was weaker than that in the CA1-CA3 region. In cerebral cortex, BREK expression was increased in the upper layers, whereas AATYK3 was expressed at similar levels across all layers.



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  		Figure 4  In situ hybridization analysis of the BREK mRNA expression. (A) Sagittal cryosections of the mouse whole brain were hybridized with cRNA probes specific for AATYK1, BREK, and AATYK3, respectively. (B) magnified views of olfactory bulb, hippocampus and cerebral cortex. (Gl, glomeruli; Mi, mitral cell; Gr, granule cell; DG, dentate gyrus; Cx, cerebral cortex).

 
Kinase activity of BREK

To confirm that BREK is a catalytically active kinase, we performed in vitro kinase assays using [{gamma}-32P] ATP. Human BREK was expressed as a FLAG-tagged protein in 293T cells, and FLAG immunoprecipitates were subjected to in vitro kinase reaction. Full-length BREK was not used in this assay, because a considerable level of phosphorylation signal was observed even with the {Delta}N mutant (420–1503) in which the putative kinase domain was deleted (data not shown). Therefore, we used the N-terminal portion of BREK encompassing amino acid residues 1–419 (WT1-419) that contains the intact kinase domain. For a negative control, we used a construct where Asp265 of BREK, which is conserved among kinases and is essential for kinase activity, was substituted to valine (DV1-419). WT1-419 but not DV1-419 underwent autophosphorylation, indicating that BREK contains a catalytically active kinase domain (Fig. 5A).



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  		Figure 5  Kinase activity of BREK. (A) FLAG-tagged partial proteins of human BREK were expressed in 293T cells. The anti-FLAG immunoprecipitates were subjected to immune-complex kinase assays using [{gamma}-32P] ATP (top). The immunoprecipitates were confirmed by blotting with anti-FLAG antibody (bottom). (B) phosphoamino acid analysis of BREK. Autophosphorylated WT1-419 shown in Figure 5 A was extracted from the gel and hydrolysed in 6 N HCl at 110 °C. The resultant mixtures were subjected to two-dimensional thin layer electrophoresis, followed by autoradiography. The positions of phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) are indicated. (C) phosphoamino acid analyses of autophosphorylated BREK family kinases. The intact kinase domains of human AATYK1 (amino acid residue 34–381), BREK (amino acid residue 94–442), and AATYK3 (amino acid residue 91–442) were expressed as GST-fusion proteins in 293T cells, purified, and the GST portions were removed by protease.

 
We then performed phosphoamino acid analysis of in vitro-phosphorylated BREK. Autophosphorylation of WT1-419 occurred predominantly at serine residues and some weak autophosphorylation of threonine was observed (Fig. 5B). Phosphoamino acid analysis of the DV1-419 kinase reaction products revealed no phosphorylated amino acids (data not shown). On the basis of these findings, we concluded that BREK is a serine/threonine kinase. AATYK1 was previously reported to have tyrosine kinase activity (Tomomura et al. 2001). To examine whether AATYK1 and AATYK3 also have serine/threonine kinase activity, we performed phosphoamino acid analysis of in vitro-phosphorylated partial proteins that contain the intact kinase domains of human AATYK1 (amino acid residues 34–381), BREK (amino acid residues 94–442), and AATYK3 (amino acid residues 91–442). These proteins were autophosphorylated predominantly at serine residues, as shown in Fig. 5C. Therefore, naming these kinases as AATYKs for apoptosis-associated tyrosine kinases does not accurately reflect their properties.

BREK is involved in NGF signalling in PC12 cells

To analyse the function of BREK in neural cells, we examined expression of BREK in various neural cell lines. PC12, NG108-15, IMR-32, SK-N-SH, B104, and P19 cells expressed BREK, whereas C6 and CG4 cells did not (data not shown). We selected PC12 cells for further analysis of BREK and examined the pattern of BREK expression after NGF stimulation. The electrophoretic mobility of endogenous BREK protein from PC12 cells was reduced within 10 min after NGF stimulation (Fig. 6A). This mobility shift was not observed when cells were pretreated with K252a, a specific inhibitor of Trk family tyrosine kinases, suggesting that activation of NGF receptor results in this mobility shift (Fig. 6A). This mobility shift was also blocked by CIAP treatment (data not shown), indicating that the NGF-induced mobility shift of BREK is due to phosphorylation. The mobility of BREK in unstimulated cells was also increased by CIAP treatment (data not shown), suggesting that BREK is phosphorylated in unstimulated PC12 cells and is further phosphorylated upon NGF stimulation.



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  		Figure 6  Roles of BREK in NGF signalling in PC12 cells. (A, B) PC12 cells were pretreated for 1 h with 100 nM K252a. (A) or 30 min with 10 µM Chelerythrine chloride (B) and then stimulated with 50 ng/ml NGF for 10 min. Total cellular proteins were subjected to Western blot analysis with anti-mouse BREK, ma4. (C) PC12 cells were treated with PDBu as indicated. Total cellular proteins were subjected to Western blot analysis with ma4. (D) PC12 cells were pretreated with or without chelerythrine (10 µM, 30 min), and stimulated with NGF (50 ng/ml, 10 min) or PDBu (10 µM, 30 min). Anti-BREK immunoprecipitates were subjected to Western blot analysis with anti-mouse BREK, ma4, or anti-phospho-(Thr) MAPK/CDK substrate antibody. Shown is the representative of four independent experiments (A–D). (E) PC12 cells were infected with retroviruses expressing mock, wild-type BREK or BREK-DV mutant. The infected cells were selected by puromycin, and then stimulated with 50 ng/ml NGF for 2 days. Total neurite length of 110–150 cells was calculated for each construct. Values represent the mean ± s.e.m. Data are from four experiments. The symbols ** and * denote statistically significant differences (P < 0.02, as determined by Student t-test for paired variables). (F) PC12 cells were infected with the indicated retroviruses. The infected cells were selected by puromycin, and then stimulated with 15 ng/ml NGF as indicated. Total cellular proteins were subjected to Western blot analysis with anti-phospho ERK (–Thr202/–Tyr204) antibodies (top), and anti-ERK1 antibodies (bottom). Shown is the representative of four independent experiments. Experiments in higher dose (50 ng/ml) showed comparable results.

 
To clarify the signalling pathway responsible for NGF-induced phosphorylation of BREK, we tested the effects of several kinase inhibitors, including PP2 for Src-family tyrosine kinases, KN-93 for Ca2+/calmodulin-dependent protein kinase II, chelerythrine for protein kinase C, wortmannin for phosphoinositide 3-kinase, and PD98059 for MEK 1/2. Chelerythrine pretreatment specifically inhibited the NGF-induced mobility shift of BREK in PC12 cells (Fig. 6B). The other inhibitors had no effect on the mobility of BREK (data not shown). These results suggest that PKC is responsible for NGF-induced phosphorylation of BREK in PC12 cells. In addition, treatment of PC12 cells with the PKC activator phorbol-12,13-dibutyrate (PDBu) induced a mobility shift in BREK (Fig. 6C). Moreover, NGF- or PDBu- induced phosphorylation of BREK was also demonstrated by immunoblotting with anti-phospho-(Thr) MAPK/CDK substrate monoclonal antibody (Cell Signalling) which specifically recognizes phosphorylated threonine prior to proline (Fig. 6D). Consistent with the mobility shift of BREK, this antibody reacted little to BREK when cells were pretreated with chelerythrine (Fig. 6D) or when immunoprecipitates were treated with CIAP (data not shown). Because the kinase activity of BREK in PC12 cells is low, it was difficult to address whether NGF stimulation and PDBu treatment alter BREK kinase activity.

NGF induces differentiation of PC12 cells into putative sympathetic neurones. To investigate the role of BREK in NGF-induced differentiation, we used a retrovirus system to express wild-type or a kinase-defective mutant (DV mutant) of human BREK in PC12 cells. After puromycin selection following retroviral infection, approximately 80% of cells were viable, whereas no non-infected control cells survived the selection. We calculated the total neurite length of the infected cells 2 days after NGF treatment. Although the number and length of neurites differed appreciably from cell to cell, which might be due to the heterogeneous nature of the PC12 cells used in our experiments, statistical analysis revealed that the DV mutant of hBREK (BREK-DV) facilitated neurite formation (Fig. 6E). Cells infected with BREK-DV viruses had significantly (P < 0.02) longer neurites than did mock-infected cells. The total neurite length of cells infected with wild-type hBREK was similar to that of mock-infected cells (Fig. 6E). These findings suggest that the kinase activity of BREK negatively regulates NGF-induced differentiation of PC12 cells and that NGF stimulation results in inactivation of downstream signalling of BREK. We could not evaluate the levels of expression of the various constructs with that of endogenous BREK because our antibodies against BREK did not cross-react between rat and human. The level of exogenous wild-type BREK was similar to that of BREK-DV (Fig. 6E). We also confirmed that the electrophoretic mobilities of exogenously expressed wild-type BREK and BREK-DV were both reduced in response to NGF or PDBu (data not shown). Thus, the shift on mobility of BREK in response to NGF appears to be due to phosphorylation of BREK by other kinase(s) and not to its autophosphorylation. Because ectopic expression of wild-type BREK had little biological effect, the level of endogenous wild-type BREK may be sufficient to permit downstream signalling of BREK.

BREK was phosphorylated rapidly (within 10 min) after NGF stimulation, and BREK-DV facilitated neurite formation. Thus, we speculated that BREK plays a role in regulation of the early biochemical signals that are important for neurite formation of PC12 cells. To address this issue, we examined whether ectopic expression of BREK alters ERK phosphorylation during the early phase of NGF signalling, which is critical for initiation of neurite formation (Qui & Green 1992). The results showed that NGF-induced (15 ng/ml) ERK phosphorylation was enhanced in cells infected with BREK-DV in comparison with mock-infected cells (Fig. 6F). The level of NGF-induced ERK phosphorylation in cells infected with wild-type hBREK was similar to that in mock-infected cells (Fig. 6F). These results were consistent with our findings that BREK-DV facilitated NGF-induced neurite outgrowth (Fig. 6E). Both BREK phosphorylation and neurite outgrowth were observed at the dose of 15 ng/ml of NGF (data not shown). Experiments with a higher dose of NGF (50 ng/ml) yielded similar results (data not shown).

Subcellular localization of BREK in PC12 cells

To examine the subcellular localization of BREK in neural cells, we performed immunofluorescence microscopy of human BREK that was retrovirally expressed in PC12 cells. BREK immunoreactivities were found throughout the cytoplasm, but were significantly concentrated at juxta-membrane regions in both differentiated and undifferentiated PC12 cells (Fig. 7A,D). In differentiated PC12 cells, BREK was also enriched at the growth cone, which was confirmed by double labelling with PKC{varepsilon} that is known to present at the growth cone in PC12 cells (Hundle et al. 1995) (Fig. 7D–F). The results were consistent with a previous report that showed presence of endogenous BREK at the growth cone of primary cortical neurones (Kesavapany et al. 2003). No immunoreactivity with anti-human BREK was detected in the mock-infected cells (Fig. 7G). BREK distribution in the growth cone suggests that BREK may be involved in local signalling that regulates neurite growth and/or axon guidance.



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  		Figure 7  Subcellular localization of BREK in PC12 cells. (A–F) PC12 cells retrovirally expressing human BREK were treated without (A–C) or with (D–F) 100 ng/ml NGF for 5 days, then the cells were double-stained with anti-human BREK antibodies, 217C (red, A and D) and anti-PKC{varepsilon} antibodies (green, B and E). Merged images are shown in C and F. The nuclei were stained with TOTO-3. Bar = 50 µm. (G) Mock-infected PC12 cells were treated with 100 ng/ml NGF for 5 days, then the cells were stained with anti-human BREK antibodies. The nuclei were stained with TOTO-3. Merged images are shown. Bar = 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In the present study, we characterized BREK, a serine/threonine kinase expressed predominantly in brain. BREK shared both sequence and structural homology with AATYK1 and AATYK3. The structures of BREK, AATYK1, and AATYK3 have several notable features that suggest their involvements in intracellular signal transduction. AATYK1 was originally described as a protein tyrosine kinase (Gazzoa et al. 1997; Tomomura et al. 2001). However, we found that AATYK1, like BREK and AATYK3, was autophosphorylated predominantly on serine residues. The data indicate that AATYK family members are serine/threonine kinases. It should be noted that in these proteins, an arginine residue, which is usually located just N-terminal to DLALRN–motif in tyrosine kinases, is changed to serine. Some serine/threonine kinases have a serine residue at the same position. Thus, the designation AATYK would be misleading in term of the characteristic of these kinases. BREK is also termed KPI-2 after Kinase/Phosphatase/Inhibitor-2 or CPRK after CDK5/p35-Regulated Kinase (Wang & Brautigan 2002; Kesavapany et al. 2003). Although complex formation of BREK with PP1C or p35 is potentially important for BREK function, the in vivo association with these putative partners or the biological significance of the association is not currently confirmed. We are afraid that these designations are also potentially misleading or do not fully represent its biological significance. AATYK3 is expressed exclusively in brain (our unpublished data), as well as AATYK1 and BREK. It could be reasonably predicted that the kinases of this family are primarily involved in brain development and/or function. Indeed, we showed that BREK negatively regulates neurite outgrowth in PC12 cells. Therefore, we believe that the designation ‘BREK’ is appropriate as compared with the other three designations.

One notable feature of BREK and this family of kinases is that all have several PXXP motifs in their C-terminal regions. PXXP motifs constitute the minimal binding site for SH3 domain-containing proteins. Several kinases with proline-rich regions are regulated by SH3-containing adaptor proteins. For example, HPK1 is activated by Grap2 (Ma et al. 2001). The proline-rich regions in BREK may be involved in regulation of its activity, intracellular localization, or substrate recognition through interactions with the SH3 domain or WW domain.

The specific tissue distribution and phosphorylation status of BREK in brain suggest that it may be involved in early postnatal brain function. We showed here that the endogenous BREK protein in PC12 cells is rapidly phosphorylated upon stimulation with NGF and that introduction of a kinase-defective mutant of BREK enhanced both ERK phosphorylation and neurite outgrowth in response to NGF. Studies with chelerythrine, a general inhibitor for PKC isozymes, suggested that BREK is phosphorylated by PKC in NGF-TrkA signalling in PC12 cells. PKCs are suggested to play roles in the differentiation of several cell types, including neurones (Goodnight et al. 1994). Phorbol esters that activate PKC stimulate the differentiation of neural cells from neuroectoderm in Xenopus embryos (Otte & Moon 1992). Phorbol esters also induce neurite outgrowth from chick sensory ganglia (Hsu et al. 1984), chick ciliary ganglion neurones (Bixby 1989), and several neuroblastoma cell lines (Pahlman et al. 1983; Spinelli et al. 1982). In addition, neurotransmitters that activate PKC may promote neurite outgrowth, which could contribute to activity-dependent remodeling of synaptic connections during normal development of the nervous system. In PC12 cells, phorbol esters enhance NGF-induced ERK phosphorylation (Roivainen et al. 1995) and neurite outgrowth (Hall et al. 1988; Roivainen et al. 1993). PC12 cells express several PKC isozymes, including those in the classical, novel, and atypical families (Wooten et al. 1994). Among these isozymes, PKC-{varepsilon} (novel) and -{zeta} (atypical) are thought to be involved in NGF-induced neurite outgrowth in PC12 cells. Over-expression of these isozymes in PC12 cells potentiates NGF-induced neurite outgrowth, whereas other isozymes, such as PKC-{alpha} and -{delta}, do not (Brodie et al. 1999; Hundle et al. 1995). In the present study, we found that PDBu treatment alone induced a shift in the mobility of BREK protein in PC12 cells, suggesting that classical and novel, but not atypical, PKC isozymes phosphorylate BREK in PC12 cells. Taken together, these data suggest that PKC-{varepsilon} may be responsible for phosphorylation of BREK in NGF-TrkA signalling in PC12 cells. Consistently, protein distribution of BREK and PKC-{varepsilon} was overlapping in PC12 cells. Importantly, mRNA expression of BREK and PKC-{varepsilon} is overlapped in brain. The spatial distribution of BREK in brain, particularly the gradient localization pattern in cerebral cortex, is also similar to that of PKC-{varepsilon} (Saito et al. 1993). In addition, both BREK and PKC-{varepsilon} are expressed predominantly in the nervous system with only trace amounts detected in non-neuronal tissues (Koide et al. 1992).

Recent studies revealed that the duration of signalling through ERKs is critical in regulation of neurite outgrowth (Qui & Green 1992), suggesting that BREK negatively regulates neurite outgrowth through inhibition of ERKs. BREK might activate negative regulators of ERKs, such as Ras-GAP, MKP1, and PP2A (Hunter 1995) during steady state in PC12 cells. Alternatively, BREK might negatively regulate the MAP kinase pathway through direct phosphorylation of Raf, MEK, or ERK. It is possible that NGF-stimulated phosphorylation down-regulates BREK activity, which results in the activation of ERKs. Over-expression of the DV mutant might also contribute to inactivation of this putative BREK-ERKs pathway. Over-expression of PKC-{varepsilon} increases NGF-induced ERK phosphorylation in PC12 cells (Brodie et al. 1999; Hundle et al. 1995), suggesting that NGF may inhibit BREK through activation of PKC. The precise mechanisms by which BREK activity is regulated in NGF-TrkA and PKC signalling need to be addressed in near future. Our findings that the kinase activity of BREK inhibits neurite outgrowth in PC12 cells contrast with those of a previous report where introduction of wild-type AATYK1 into SHSY-5Y cells induced neurite outgrowth (Raghunath et al. 2000). Because BREK/KPI-2 phosphorylates PP1C (Wang & Brautigan 2002) that may be involved in actin organization (MacMillan et al. 1999), BREK may control neurite extension by acting on PP1C. Further experiments to clarify the distinct roles of BREK, AATYK1, and AATYK3 are needed to resolve this inconsistency.

In conclusion, we identified BREK, a serine/threonine kinase expressed predominantly in brain, and biochemically characterized BREK by examining its temporal and spatial expression and kinase activity. We show that BREK was phosphorylated after NGF stimulation through the TrkA-PKC pathway and suggested that its kinase activity modulates ERK activity and neurite outgrowth of PC12 cells.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Reagents

Anti-FLAG monoclonal antibody (M2), chelerythrine chloride, phorbol-12, 13-dibutyrate (PDBu), wortmannin, poly L-lysine, and K252a were from Sigma Chemical Co. Calf intestine alkaline phosphatase was from Roche Diagnostics. NGF was from TaKaRa Biomedicals. PD98059, KN-93, and PP2 were from calbiochem. Anti-phospho ERK (–Thr202/–Tyr204) polyclonal antibodies and anti-phosoho-(Thr) MAPK/CDK substrate monoclonal antibody were from Cell Signalling. Anti-ERK1 polyclonal antibodies (C-16) were from Santa Cruz Biotechnology. Goat anti-rabbit IgG-Alexa488-conjugated antibody, TOTO-3, and Zenon Alexa Fluor 546 Rabbit IgG labelling kit were from Molecular Probes. Rabbit anti-PKC{varepsilon} polyclonal antibodies were a gift from Dr K. Chida (University of Tokyo, Japan).

Cell lines

293T, P19 (mouse embryonal carcinoma), B104 (rat neuroblastoma), IMR-32 (human neuroblastoma), SK-N-SH (human neuroblastoma), NG108-15 (somatic cell hybrid of rat glioma and mouse neuroblastoma), and C6 (rat glioma) cells were cultured according to the recommendations of American Type Culture Collection. CG4 (rat oligodendroglia) cells were cultured as previously described (Espinosa de los Monteros & de Vellis 1994). PC12 (rat pheochromocytoma) cells were grown in DMEM supplemented with 10% horse serum (Invitrogen) and 5% foetal bovine serum (Invitrogen) in BIOCOAT Collagen I Cellware plates (Becton Dickinson Labware).

Plasmid construction

The human BREK cDNA (KIAA1079, GENBANK Accession No. AB029002) clone was a kind gift of the Kazusa DNA Research Institute (Chiba, Japan). Site-directed mutagenesis was accomplished by a modified overlap extension method (Tomomura et al. 2001) using PCR with Pfx DNA polymerase (Invitrogen). The nucleotide sequences of mutants were confirmed by bi-directional sequencing. The cDNAs were cloned into the expression vector pIRES-hrGFP-1a (Stratagene) to express C-terminal FLAG-tagged proteins (Figs 2A,B and 5A,B) or pME-GST (a derivative of pME-18S) to express GST-fusion proteins (Fig. 5C) in mammalian cells. For retroviral transfer, a full-length cDNA encoding wild-type or DV mutant BREK was cloned into the expression vector pMX-puro (Suzuki et al. 2002). To generate rabbit anti-BREK polyclonal antibodies, expression plasmids for glutathione S-transferase (GST)-BREK were constructed by inserting mouse cDNA fragments that encode portions of mouse BREK corresponding to amino acid residues 766–876 (for ma2) and 1121–1200 (for ma4) of human BREK and a human cDNA fragment encoding amino acid residues 1387–1454 of human BREK (for 217C) into the pGEX-5X-1 expression vector (Amersham Biosciences).

Analysis of BREK mRNA expression

For Northern blot analysis, total RNAs (10 µg) prepared from different mouse tissues were blotted on to a nylon membrane (Hybond-N, Amersham Biosciences) and subjected to hybridization with a 32P-labelled cDNA probe. To prepare the probe, a 250-bp fragment of mouse EST sequence (nucleotides 116–365, GENBANK Accession No. AI747891, corresponding to 5'-portion of the mouse BREK mRNA) was amplified by PCR with primers 5'-ATGCTCCTGCTGCTCCTGCT-3' and 5'-TGTCTTCTGCTGGTGGGGTG-3'. The membrane was incubated for 3 h at 65 °C in 7% SDS and 0.5 M sodium phosphate buffer (pH 8.0) and then for 16 h at 65 °C in the same buffer containing the 32P-labelled cDNA probe. After hybridization, the membrane was washed in 0.1 x SSC and 0.1% SDS at 50 °C. In situ hybridization was performed with [{alpha}-35S] UTP-labelled cRNA probe (Yoshida et al. 2000). Partial mouse cDNA fragments for and (corresponding to amino acid residues 716–834 of human and 947–1067 of human , respectively) were obtained by RT-PCR, verified by sequencing, and then used as probes.

Antibodies against BREK

Expression of GST-BREK fusion proteins in E. coil BL21-strain was induced with isopropyl-ß-D-thiogalactopyranoside, and the proteins were purified with glutathione-Sepharose (Amersham Biosciences) as previously described (Fujimoto et al. 1999). New Zealand White rabbits were immunized with purified GST-BREK fusion proteins. Antibodies raised against BREK were purified with HiTrap NHS-activated affinity columns coupled with GST-BREK proteins (Amersham Biosciences).

Transfection and retroviral infection

293T cells were transfected with expression plasmids by the calcium phosphate method as described (Fujimoto et al. 1999). Retroviral infection of PC12 cells was performed as described (Brodie et al. 1999) with pMX-puro vector.

Protein preparation, immunoprecipitation, and immunoblotting

Protein lysates were prepared with lysis buffer (1% NP-40, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulphonyl fluoride, 1 mM NaF, 1 mM Na3VO4). Insoluble materials were removed by centrifugation (12 000 g, 10 min, 4 °C). The lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene diflouride membranes (Trans-Blot, Bio-Rad). The membranes were probed with 1 µg/ml antibodies, and signals were visualized by chemiluminescence (Renaissance, NEN Life Sciences). For immunoprecipitation, cell lysates were precleared with Protein A-Sepharose 4 Fast Flow (Amersham Biosciences) for 1 h at 4 °C. The precleared lysates were incubated for 2 h at 4 °C with antibodies indicated and Protein A-Sepharose. The immune complex was washed four times with TNE buffer (1% NP-40, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA).

Kinase reaction and phosphoamino acid analysis

The BREK family proteins were prepared as anti-FLAG immunoprecipitates from transfected 293T cells or expressed as GST-fusion proteins in 293T cells, purified, and treated with PreScissionTM Protease (Amersham Biosciences) to remove GST portions. These proteins were incubated with [{gamma}-32P] ATP (370 kBq, 110 TBq/mmol) in kinase buffer (20 mM HEPES (pH 7.4), 10 mM MgCl2, and 10 mM MnCl2) for 30 min at 37 °C. The reaction mixtures were analysed by SDS-polyacrylamide gel electrophoresis. Phosphoamino acid analysis of the phosphorylated proteins was performed as previously described (Boyle et al. 1991).

NGF and PDBu stimulation of PC12 cells

For the analysis of BREK phosphorylation, PC12 cells (1 x 105) were pretreated with or without kinase inhibitors KN-93 (10 µM, 30 min), chelerythrine (10 µM, 30 min), PD98059 (100 µM, 1 h), wortmannin (100 nM, 30 min), PP2 (30 µM, 1 h), or K252a (100 nM, 1 h), and stimulated with NGF (50 ng/ml, 10 min) or PDBu (as indicated in Fig. 6C,D). For analysis of neurite extension, PC12 cells infected with Moloney murine leukaemia virus expressing a puromycin-resistance gene were selected with puromycin (0.8 µg/ml) for 48 h. The selected cells (5 x 104) were plated in 6-well plates, and stimulated with NGF (50 ng/ml) for 2 days. The neurite length of the cells was calculated with TI-Workbench (computer software provided by Dr T. Inoue, University of Tokyo, Japan). Student t-test for paired variables was used to examine differences elicited by NGF treatment, and data were considered significantly different at P < 0.05. The cells were also stimulated with NGF (15 ng/ml) to evaluate ERK activation.

Subcellular localization of BREK in PC12 cells

PC12 cells infected with mock or wild-type BREK-harbouring viruses (8 x 103 cells/cm3) were plated on slides coated with poly L-lysine. After 5 days of culture with or without 100 ng/ml NGF, the cells were fixed with 4% paraformaldehyde/PBS, permeablized with 0.2% Triton X-100/PBS, and then incubated with the blocking solution (1% BSA, 0.1 mg/ml RNaseA/PBS). For double staining, the cells were incubated with rabbit anti-PKC{varepsilon} antibodies (1 µg/ml) and TOTO-3, followed by staining with goat anti-rabbit IgG-Alexa488-conjugated antibody. After re-fixation, the cells were incubated with rabbit anti-human BREK antibodies 217C (1 µg/ml), which were prelabelled with Zenon Alexa Fluor 546 Rabbit IgG labelling kit. Immunofluorescence was observed using a Radiance 2100 (Bio-Rad).


   Acknowledgements
 
We thank Dr T. Inoue for TI-Workbench, Dr K. Chida for Rabbit anti-PKC{varepsilon} polyclonal antibodies, Dr Y. Abe and Mr K. Yokoyama for valuable discussion. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Shigekazu Nagata

* Correspondence: E-mail: tyamamot{at}ims.u-tokyo.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 13 October 2003
Accepted: 11 December 2003




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