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Genes to Cells (2008) 13, 949-964. doi:10.1111/j.1365-2443.2008.01219.x
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Palmitoylation-dependent endosomal localization of AATYK1A and its interaction with Src

 Koji Tsutsumi1,*, Mineko Tomomura2, Teiichi Furuichi3 and Shin-ichi Hisanaga1,*

1 Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan
2 Laboratory for Neuronal Growth Mechanisms, and
3 Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Apoptosis-associated tyrosine kinase 1 (AATYK1), also named LMTK1, was previously isolated as an apoptosis-related gene from 32Dcl3 myeloid precursor cells, but its precise function remains unknown. AATYK1A, an isoform without a transmembrane domain, is highly expressed in neurons. We identified palmitoylation of AATYK1A at three N-terminal cysteine residues in cortical cultured neurons and COS-7 cells and found that palmitoylation determined localization of AATYK1A to the transferrin receptor-positive recycling endosomes. Further, we identified the tyrosine kinase Src as a novel AATYK1A-interacting protein. Src and Fyn phosphorylated AATYK1A at tyrosines 25 and 46 in a palmitoylation-dependent manner. The association of AATYK1A with Src in endosomes was also found to be palmitoylation-dependent. These results indicate that palmitoylation is a critical factor not only for the subcellular localization of AATYK1A but also for its interaction with Src.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Protein phosphorylation is the most common post-translational modification in eukaryotic cells that can reversibly regulate protein function. The human genome contains approximately 500 protein kinase genes (Manning et al. 2002), and although many protein kinases have been studied extensively, there are still many that remain to be characterized, including apoptosis-associated tyrosine kinases 1-3 (AATYK1-3), also called lemur tyrosine kinases 1-3 (LMTK1-3). AATYK1 was originally identified as an up-regulated gene during apoptosis following differentiation of 32Dcl3 myeloid precursor cells by interleukin-3 deprivation (Gaozza et al. 1997). AATYK2 and AATYK3 were identified as AATYK1 family kinases by searching for tyrosine kinases in the human genome (Robinson et al. 2000). Several groups independently isolated AATYK2 (LMTK2) as protein phosphatase 1 inhibitor-2 binding protein (KP1-2) (Wang & Brautigan 2002), cyclin-dependent kinase 5 (Cdk5) activator p35 binding protein (Cprk) (Kesavapany et al. 2003) and a novel kinase through a database search (Brek) (Kawa et al. 2004). Members of the AATYK/LMTK family are large proteins composed of 1300–1500 amino acids that consist of an N-terminal tyrosine kinase-like domain and a C-terminal proline-rich long tail region. Although AATYKs have been predicted to be tyrosine kinases based on kinase domain amino acid sequence similarity with receptor tyrosine kinases, serine/threonine kinase activity has mainly been reported (Wang & Brautigan 2002, 2006; Kawa et al. 2004).

AATYK1 is highly expressed in the nervous system (Gaozza et al. 1997; Baker et al. 2001; Tomomura et al. 2001, 2003) and is up-regulated during retinoic acid-induced neuronal differentiation of P19 embryonal carcinoma cells (Baker et al. 2001). Expression of AATYK1 is increased in cultured cerebellar granule cells undergoing apoptosis induced by low KCl (Tomomura et al. 2001). Further, over-expression of AATYK1 promotes neurite outgrowth and stimulates low KCl-induced apoptosis of cerebellar granule cells (Tomomura et al. 2003; Tomomura & Furuichi 2005). These results implicate AATYK1 in neuronal differentiation as well as in apoptosis. AATYK1 has been shown to interact with several signaling proteins such as Cdk5 activator p35 (Honma et al. 2003), protein phosphatase 1 (PP1) and Ste-20-related proline-alanine-rich kinase (SPAK) (Gagnon et al. 2007). However, the physiological relevance of these interactions has not yet been fully characterized. Further, in addition to the initial identification of AATYK1 as a cytoplasmic protein (referred to as AATYK1A hereafter), a splicing variant of AATYK1 containing a transmembrane domain was reported (referred to as AATYK1B hereafter) (Baker et al. 2001; Tomomura et al. 2007). The functional differences between AATYK1A and AATYK1B have not been addressed.

Palmitoylation is another reversible post-translational protein modification, which involves addition of palmitate, a 16-carbon saturated fatty acid, to specific cysteine residues through a thioester linkage (Resh 1999; El-Husseini & Bredt 2002; Bijlmakers & Marsh 2003). In neurons, protein palmitoylation regulates many cellular activities such as the trafficking of signaling molecules, neurotransmitter receptors and synaptic scaffolding proteins (Huang & El-Husseini 2005). For example, postsynaptic density protein-95 (PSD-95) requires palmitoylation for postsynaptic targeting and clustering (Craven et al. 1999), and palmitoylation of PSD-95 inhibits {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor internalization (El-Husseini et al. 2002). Palmitoylation also regulates presynaptic membrane dynamics, such as SNAP-25-mediated disassembly of the SNARE complex (Vogel et al. 2000), presynaptic targeting of GABA-synthesizing enzyme GAD65 (Kanaani et al. 2004) and presynaptic trafficking of calcium sensor synaptotagmin I (Kang et al. 2004).

AATYK1A has been suggested to be a palmitoylated protein (Tomomura et al. 2007). Therefore, we examined the palmitoylation of AATYK1A and its role in AATYK1A subcellular localization. We identified palmitoylation of AATYK1A at cysteines 4, 6 and 7; palmitoylation of AATYK1A at any of these cysteines mediated its co-localization with transferrin receptor–positive endosomes in COS-7 cells and neurons. We found that Src and Fyn, members of the Src family tyrosine kinases, interact with AATYK1A. Src and Fyn phosphorylated AATYK1A at tyrosines 25 and 46 and Src associated with AATYK1A in a palmitoylation-dependent manner. These results suggest that palmitoylation plays critical roles in the subcellular localization of AATYK1A and interactions with Fyn or Src tyrosine kinases.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Palmitoylation of AATYK1 in neurons and COS-7 cells

AATYK1A, an isoform of AATYK1 expressed in mammalian brains, was recently suggested to bind to membranes via palmitoylation (Tomomura et al. 2007). However, palmitoylation has not yet been verified experimentally; thus, we set out to confirm that palmitoylation of AATYK1 occurs in cultured neurons. AATYK1 was found in the membrane fraction of a cultured cortical neuron homogenate, and a portion was solubilized when neurons were treated with 2-bromopalmitate, an inhibitor of palmitoylation (Fig. 1A). A part of PSD-95, a palmitoylated protein, was also detected in the soluble fraction after 2-bromopalmitate treatments, but N-methyl-D-aspartate receptor 2A (NR2A), an integral membrane protein, rema-ined in the membrane fraction. To test whether AATYK1 is indeed palmitoylated, neurons were cultured in the presence of [3H]palmitate for 8 h, and palmitoylation of AATYK1 was analyzed by autoradiography after immunoprecipitation (Fig. 1B). AATYK1 was labeled with [3H]palmitate as well as the positive control PSD-95, indicating that endogenous AATYK1 is palmitoylated in neurons.


Figure 1
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  		Figure 1  Palmitoylation of AATYK1A. (A) Mouse brain cortical neuron cultures at 12 DIV were incubated with (+) or without (–) 100 µM 2-bromopalmitate (2-BP) for 8 h, and their homogenates were separated into soluble (S) and particulate (P) fractions by centrifugation. Proteins were detected by immunoblotting with anti-AATYK1, anti-PSD-95, and anti-NR2A. PSD-95 is a cytoplasmic protein bound to membranes via palmitoylation, and NR2A is an integral membrane protein. (B) Cortical neurons at 12 DIV were cultured in the presence of [3H]palmitate for 8 h, and then palmitoylation of AATYK1 or PSD-95 was detected by autoradiography after immunoprecipitation with anti-AATYK1 or anti-PSD-95 (right panels). Their immunoprecipitation was confirmed by immunoblotting with anti-AATYK1 or anti-PSD-95 (left panels). (C) The amino acid sequence of the N-terminal region of AATYK1A compared with that of N-terminal palmitoylated proteins PSD-95, GAP-43 and AKAP18. Cysteine residues that are possible or known palmitoylation sites are indicated in gray. (D) COS-7 cells expressing N390, the N-terminal 390-residue fragment of AATYK1A tagged with Myc at the C terminus, were incubated with (+) or without (–) 100 µM 2-BP for 4 h, and then the soluble and particulate fractions are separated as described in (A). N390 was detected by immunoblotting with anti-Myc (9E10). β-tubulin (Tub) was used as a cytosolic protein marker. (E) N390 or its cysteine-to-alanine mutant (C4/6/7A) was transfected into COS-7 cells, and then their distribution in the soluble and particulate fractions was examined by immunoblotting. (F) N390 or N390-C4/6/7A expressed in COS-7 cells was metabolically labeled with [3H]palmitate for 4 h and was immunoprecipitated with anti-Myc. Palmitoylation was detected by autoradiography after SDS-PAGE. Their immunodetection is shown in the lower panel.

 
We next determined the location of the palmitoylation sites in AATYK1A. AATYK1A contains three cysteine residues at positions 4, 6 and 7 in the N-terminal region, similar to other palmitoylated proteins such as PSD-95, GAP-43 and AKAP18 (Resh 1999) (Fig. 1C). The palmitoylation site prediction algorithms, CSS-Palm (http://bioinformatics.lcd-ustc.org/css_palm/ [Zhou et al. 2006]) and NBA-Palm (http://www.bioinfo.tsinghua.edu.cn/NBA-Palm/index [Xue et al. 2006]), predicted that these cysteines could be palmitoylated (Supporting Information Table SI). To test whether these sites were in fact palmitoylated, we expressed an N-terminal fragment of AATYK1A, N390 (amino acid residues 1-390) and a mutant of N390 in which all three putative palmitoylation site cysteines were changed to alanine (N390-C4/6/7A). N390 expressed in COS-7 cells was recovered in the membrane fraction after centrifugation; however, as with the full-length AATYK1A (Fig. 1A), it was found in the supernatant when cells were treated with 2-bromopalmitate (Fig. 1D). By contrast, N390-C4/6/7A was recovered in the soluble fraction even in the absence of 2-bromopalmitate (Fig. 1E). N390 was labeled with [3H]palmitate, whereas N390-C4/6/7A was not (Fig. 1F). These results indicate that palmitoylation of AATYK1A occurs on the N-terminal cysteine residues at positions 4, 6 and 7 and regulates association of AATYK1A with membranes.

Effect of palmitoylation on subcellular localization of AATYK1A

We examined the effect of palmitoylation on the intracellular localization of AATYK1A by using cysteine-to-serine mutants of N390 expressed in COS-7 cells. N390 localized in the perinuclear region (Fig. 2A), and when the cells were treated with 2-bromopalmitate, the N390 distribution became diffuse in whole cells (Fig. 2B). The C4/6/7S mutant, in which the cysteine residues 4, 6 and 7 were replaced with serine, also showed a diffuse distribution (Fig. 2C), suggesting that palmitoylation is required for perinuclear localization of N390.


Figure 2
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  		Figure 2  Effect of N-terminal cysteine mutations on subcellular localization of the N390 fragment of AATYK1A. COS-7 cells expressing N390 (A, B) or various cysteine-to-serine mutants (C–I) were fixed and then stained with anti-Myc (9E10), followed by Alexa488-conjugated secondary antibody. Effect of 100 µM 2-BP on the distribution of N390 is shown in (B). Cysteine-to-serine mutants are as follows: (C), triple mutant C4/6/7S; (D)–(F), double mutant C4/6S, C4/7S and C6/7S; (G)–(I), single mutant C4S, C6S and C7S. Arrowheads in (G)–(I) indicate accumulation at the perinuclear region. Scale bar, 10 µm.

 
To elucidate which cysteine residues are necessary for accumulation in the perinuclear region, we generated double (C4/6S, C4/7S and C6/7S) and single (C4S, C6S and C7S) mutants of the three cysteine residues and observed their distribution in COS-7 cells. All of the double mutants were distributed diffusely in the cytoplasm (Fig. 2D–F), whereas all of the single mutants showed perinuclear accumulation with some diffuse cytoplasmic distribution (Fig. 2G–I). These results indicate that accumulation of AATYK1A in the perinuclear region increases with the number of palmitoylated residues and requires palmitoylation on at least two cysteines among the three possible sites.

Localization of AATYK1A in transferrin receptor–positive endosomes

Full-length AATYK1A expressed in COS-7 cells showed perinuclear accumulation similar to N390, although the punctate staining tended to spread throughout the entire cytoplasm (Fig. 3A). Because the Golgi is located in the perinuclear region, we examined whether AATYK1A co-localized with the Golgi using antibodies against GM130 and Golgin-97 as cis-Golgi and trans-Golgi markers, respectively (Fig. 3A–F). Although AATYK1A localized at the perinuclear region close to the Golgi, staining of AATYK1A did not coincide with that of GM130 (insets of Fig. 3A–C) and only partially coincided with that of Golgin-97 (insets of Fig. 3D–F). We next examined localization of AATYK1A in recycling endosomes, which also are located in the perinuclear region (Yamashiro et al. 1984), using the transferrin receptor (TfR) as a marker (Fig. 3G–I). Punctate staining of AATYK1A in the perinuclear region agreed well with that of TfR (Fig. 3J–L). A portion of vesicular staining of AATYK1A in the cell periphery also overlapped with TfR staining (Fig. 3M–O). Similar results were obtained with HeLa cells (Supporting Information Fig. S1A). N390 expressed in COS-7 cells also co-localized with TfR (Supporting Information Fig. S1B). These data indicate that AATYK1A expressed in COS-7 cells largely localizes to TfR-positive endosomes.


Figure 3
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  		Figure 3  Localization of AATYK1A in transferrin receptor–positive endosomes. COS-7 cells were transiently transfected with full-length AATYK1A, and after 24 h transfection cells were doubly labeled with anti-Myc for AATYK1A (A, D, G) and either anti-GM130 as a cis-Golgi marker (B), anti-Golgin-97 as a trans-Golgi marker (E), or anti-transferrin receptor (TfR) as a recycling endosome marker (H). Their merged images are shown in (C), (F), and (I). Insets in (A)–(F) are higher magnifications of perinuclear staining, indicated by dotted rectangles. Higher magnifications of the perinuclear (PN) and cell peripheral region (CP) indicated by dotted rectangles 1 and 2, respectively, in (G)–(I) are shown in (J)–(L) and (M)–(O). Scale bars, 10 µm in (A), (D) and (G), 2 µm in (J) and (M).

 
Localization of endogenous AATYK1 to endosomes in cultured neurons

To examine whether endogenous AATYK1 localizes to endosomes, cultured cortical neurons whose recycling endosomes were labeled with Alexa-transferrin were immunostained with anti-AATYK1. Alexa-transferrin was detected in a punctate pattern in the perinuclear region and in neurites (Fig. 4A, b,e). Endogenous AATYK1 also showed punctate staining in the soma and neurites (Fig. 4A, a,d). Merged images show that a part of AATYK1 clearly co-localized with Alexa-transferrin (Fig. 4A, c,f). In contrast, AATYK1 did not co-localize with the Golgi, which was stained with anti-GM130 (Supporting Information Fig. S1C). We also confirmed association of AATYK1 with endosomes biochemically. A homogenate of cultured neurons was separated into several membrane fractions by differential centrifugation. AATYK1 was predominantly recovered in the postsynaptic membrane LP1 and high-density microsomal HDM fractions, the identical distribution of TfR (Fig. 4B). These data indicate that endogenous AATYK1 localizes to recycling endosomes in neurons.


Figure 4
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  		Figure 4  Endosomal localization of AATYK1 in cultured neurons. (A) Cultured cortical neurons at 15 DIV were incubated with 20 µg/mL Alexa546-conjugated transferrin for 2 h and labeled with anti-AATYK1. The AATYK1 staining (a and d) co-localized with internalized Alexa-transferrin (Alexa-Tfn) (b and e) in the cell body (a–c) and neurites (d–f). Merged images are shown in (c) and (f). Punctate co-staining in neurites is indicated by white asterisks (d–f). Scale bars represent 10 µm in (a) and 5 µm in (d). (B) Cortical neuron culture extracts were separated into several subcellular fractions: H, homogenate; LP1, synaptosomal membrane fraction; LP2, synaptic vesicle–enriched fraction; HDM, high-density microsome fraction; LDM, low-density microsome fraction. The same amount of protein (5 µg) was applied to each lane. The blots were probed with anti-AATYK1, anti-TfR, anti-PSD-95, anti-NR2A, anti-synaptotagmin, and anti-dynamin. PSD-95 and NR2A were used as postsynaptic markers, and synaptotagmin and dynamin were presynaptic markers.

 
Palmitoylation is necessary for endosomal targeting of AATYK1A in COS-7 and cultured neurons

AATYK1A expressed in COS-7 cells co-localized with TfR. To investigate the role of palmitoylation in endosomal localization of AATYK1A, COS-7 cells or cultured cortical neurons expressing AATYK1A or AATYK1A-C4/6/7S were incubated with Alexa-transferrin (Fig. 5). In COS-7 cells, AATYK1A co-localized with internalized Alexa-transferrin in the perinuclear region (Fig. 5A, a–c and their insets). AATYK1A-C4/6/7S was distributed diffusely throughout the cytoplasm and did not co-localize with Alexa-transferrin (Fig. 5A, d–f and insets). Similar results were obtained with neurons; AATYK1A showed punctate staining, which coincided with Alexa-transferrin in the soma and neurites (Fig. 5B, a–c and insets), and AATYK1A-C4/6/7S displayed diffuse staining throughout the soma and neurites (Fig. 5B, d–f and insets). These results indicate that AATYK1A is targeted to recycling endosomes in both COS-7 cells and neurons by palmitoylation of cysteines at positions 4, 6 and 7.


Figure 5
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  		Figure 5  Palmitoylation is required for endosomal targeting of AATYK1A. (A) COS-7 cells expressing AATYK1A (a–c) or AATYK1A-C4/6/7S (d–f) were incubated with 5 µg/mL Alexa546-conjugated transferrin (Tfn) for 1 h. AATYK1A was stained with anti-Myc (9E10) followed by Alexa488-conjugated secondary antibody (a and d). Alexa-Tfn is shown in (b) and (e). Insets show a higher magnification view of the perinuclear region indicated by dotted rectangles. Scale bars, 10 µm. (B) AATYK1A (a–c) or AATYK1A-C4/6/7S (d–f) was transfected into cortical cultured neurons at 6 DIV; 46 h after transfection cells were incubated with 20 µg/mL Alexa546-conjugated transferrin (Tfn) for 2 h. AATYK1A was detected by anti-Myc staining (a and d). Alexa-Tfn is shown in (b) and (e). Co-staining in a neurite is indicated by white arrows in (a)–(c). Scale bars, 10 µm.

 
Phosphorylation of AATYK1A on tyrosine is dependent on palmitoylation

AATYK1A expressed in HEK293 cells is phosphorylated at tyrosine residues; this phosphorylation is decreased in the kinase deficient mutant (Tomomura et al. 2001). We examined the effect of palmitoylation on tyrosine phosphorylation of AATYK1A by immunoblotting with antiphosphotyrosine. Immunoreaction with the anti-phosphotyrosine was detected with AATYK1A but not with AATYK1A-C4/6/7S (Fig. 6A). Tyrosine phosphorylation of AATYK1A was significantly enhanced by treatment of cells with pervanadate, an inhibitor of tyrosine phosphatase (Fig. 6B, a). To test whether this pervanadate-enhanced phosphorylation was dependent on palmitoylation of AATYK1A, we assessed tyrosine phosphorylation of AATYK1A-C4/6/7S in COS-7 cells treated with pervanadate. Phosphorylation of AATYK1A-C4/6/7S was remarkably decreased (Fig. 6B, b), indicating that palmitoylation stimulates tyrosine phosphorylation of AATYK1A. To identify the phosphorylation sites, we generated several deletion mutants and examined their tyrosine phosphorylation in COS-7 cells treated with pervanadate (Supporting Information Fig. S2). Pervanadate increased tyrosine phosphorylation of N390 (Fig. 6C, a) and the phosphorylation was not observed in N390-C4/6/7A (Fig. 6C, b). Interestingly, a fragment of AATYK1A lacking the kinase domain ({Delta}KD) was still strongly phosphorylated on tyrosine (Fig. 6D). These results indicate that the tyrosine phosphorylation site exists in the N-terminal 390 residues, outside of the kinase domain, and that the pervanadate-enhanced tyrosine phosphorylation is not autocatalytic. There are 14 tyrosine residues in N390, but only two of them, tyrosines 25 and 46, are present outside of the kinase domain. To determine which of these tyrosines is phosphorylated, we mutated one or both tyrosine residues in N390 to phenylalanine (Fig. 6E). Mutation at tyrosine 25 or 46 decreased tyrosine phosphorylation, and the double mutant N390-Y25/46F was not phosphorylated, consistent with the result that the AATYK1A deletion mutant composed of residues 78–667 (M78-667), lacking tyrosines 25 and 46, was not phosphorylated (Supporting Information Fig. S2). These results indicate that both tyrosines 25 and 46 in the N terminus of AATYK1A are sites for phosphorylation.


Figure 6
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  		Figure 6  Palmitoylation stimulates tyrosine phosphorylation of AATYK1A. (A) AATYK1A or AATYK1A-C4/6/7S was expressed in COS-7 cells. Tyrosine phosphorylation of AATYK1A was assessed by immunoblotting with anti-phosphotyrosine (pY) after immunoprecipitation with anti-AATYK1. (B) COS-7 cells expressing AATYK1A were treated with 100 µM pervanadate, a tyrosine phosphatase inhibitor, for the indicated times (a). Tyrosine phosphorylation of AATYK1A or AATYK1A-C4/6/7S expressed in COS-7 cells was examined after 100 µM pervanadate treatment for 10 min (b). Phosphotyrosine immunoblotting was carried out after immunoprecipitation with anti-Myc or anti-AATYK1A. (C) COS-7 cells expressing N390 were treated with 100 µM pervanadate for the indicated times (a). Tyrosine phosphorylation of N390 or N390-C4/6/7A expressed in COS-7 cells was examined after 100 µM pervanadate treatment for 10 min (b). Phosphotyrosine immunoblotting was carried out after immunoprecipitation with anti-Myc. (D) Tyrosine phosphorylation of N667 and its kinase domain deletion mutant ({Delta}KD) in the presence or absence of 100 µM pervanadate was examined by immunoblotting with anti-phosphotyrosine and for protein expression by immunoblotting with anti-Myc. (E) Putative tyrosine phosphorylation sites in N390 (upper panel). N390 or its tyrosine-to-phenylalanine mutants, Y25F, Y46F and Y25/46F, was expressed in COS-7 cells, and tyrosine phosphorylation was analyzed by immunoblotting with anti-phosphotyrosine (pY) (lower panel).

 
Src phosphorylates and associates with AATYK1A

We next investigated the tyrosine kinase that phosphorylates AATYK1A. We tested Src family tyrosine kinases (SFKs) as candidates because they are also myristoylated or palmitoylated proteins known to be involved in membrane trafficking (Brown & Cooper 1996). COS-7 cells expressing N390 were treated with the SFK inhibitor PP2, and phosphorylation of N390 was examined (Fig. 7A). Pervanadate-enhanced tyrosine phosphorylation of N390 was suppressed in the presence of PP2 but not in the presence of its inactive analogue PP3, indicating that a member of the SFKs catalyzes pervanadate-enhanced phosphorylation. To test whether Fyn or Src, members of the SFKs expressed in neurons (Thomas & Brugge 1997), phosphorylates AATYK1A, we co-expressed a constitutively active form (Fyn-Y531F or Src-Y530F) along with N390 in COS-7 cells. N390 was phosphorylated at tyrosine residues by co-expression with Fyn-Y531F or Src-Y530F even in the absence of pervanadate (Fig. 7B). To examine if AATYK1A is directly phosphorylated by SFKs, we assessed whether the N-terminal 1–78 residues of AATYK1A tagged with GST (GST-N78), which contained tyrosines 25 and 46 as the sole tyrosine residues, could be phosphorylated in vitro by purified Fyn-Y531F. Fyn-Y531F effectively phosphorylated GST-N78 (Fig. 7C). These data indicate that Fyn, and probably Src, directly phosphorylate AATYK1A at tyrosines 25 and 46.


Figure 7
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  		Figure 7  Phosphorylation and association of AATYK1A with Src. (A) COS-7 cells expressing N390 were treated with 10 µM PP2 or PP3 for 1 h and with 100 µM pervanadate for an additional 5 min. Tyrosine phosphorylation of N390 was assessed by immunoblotting with anti-phosphotyrosine (pY) after immunoprecipitation with anti-Myc. (B) N390 was co-expressed with the constitutively active form of Fyn (Fyn-Y531F) or the constitutively active form of Src (Src-Y530F) in COS-7 cells. Tyrosine phosphorylation of N390 was examined using anti-phosphotyrosine after immunoprecipitation with anti-Myc. Expression of Fyn-Y531F or Src-Y530F is shown by immunoblotting of the lysate with anti-Fyn or anti-Src. (C) GST-AATYK1A-N78 (GST-N78) or GST was incubated in the presence of [{gamma}-32P]ATP with Fyn-Y531F prepared from COS-7 cells by immunoprecipitation. Phosphorylation was detected by autoradiography after SDS-PAGE (left panel). Right panel shows Coomassie Brilliant Blue (CBB) staining of proteins. (D) Brains of 6-week-old mice were homogenized, and subcellular fractionation was carried out by differential centrifugation as described in the Experimental procedures. Src, AATYK1 and TfR were detected in these fractions by immunoblotting with anti-Src, anti-AATYK1 and anti-TfR, respectively. The same amount of protein (5 µg) was applied to each lane. (E) Co-immunoprecipitation of Src with anti-AATYK1. AATYK1 was immunoprecipitated with anti-AATYK1 from the P3 microsome fraction in (D) suspended in buffer containing 1% Nonidet P-40. (F) Direct association between AATYK1A and Src. AATYK1A from COS-7 cells was purified by Ni-NTA column (Qiagen) from COS-7 cells and mixed with Src prepared from COS-7 cells by anti-Src antibody bound to protein G beads. The association of AATYK1A with Src was analyzed by immunoblotting with anti-AATYK1. (G) Src was co-expressed with either AATYK1A or the non-palmitoylation mutant AATYK1A-C4/6/7S in COS-7 cells. Their association was analyzed by co-immunoprecipitation with anti-AATYK1 (upper panel). The amount of Src co-immunoprecipitated with AATYK1A or AATYK1A-C4/6/7S was measured using Quantity One (Bio-Rad) (lower panel). Results are presented as the mean ± SE (n = 4; *P < 0.005, Student's t-test). (H) COS-7 cells expressing both AATYK1A and Src were incubated with 5 µg/mL Alexa546-conjugated transferrin for 1 h, and then double-stained with anti-AATYK1 (a) and anti-Src (b). Alexa-transferrin is shown in (c) (Alexa-Tfn). (e)–(h) is higher magnifications of the perinuclear region indicated by dotted rectangles in (a)–(d). Co-localization of the three proteins is represented by white color in merged images (d and h). Scale bars, 10 µm in (a) and 2 µm in (e).

 
AATYK1 localized in recycling endosomes in neurons (Fig. 4). Because Src localizes in endosomes (Kaplan et al. 1992), we next examined the physical association between AATYK1 and Src. To test for the association of AATYK1 and Src in brains, we first fractionated endosomes from adult mouse brain homogenates by differential centrifugation. Endosomes, which are marked by TfR, were mainly recovered in the P3 microsome fraction (Fig. 7D), in which both AATYK1 and Src signals were strongly detected. To observe the possible association between AATYK1 and Src, we examined whether Src could be co-immunoprecipitated from the microsome fraction with AATYK1. Src was specifically detected in the immunoprecipitates with anti-AATYK1 but not with control IgG (Fig. 7E), indicating that a fraction of Src associates with AATYK1 in mouse brain endosomes.

To investigate whether Src associated with AATYK1 via a direct interaction, we carried out in vitro binding assay. AATYK1A, which was purified from COS-7 cells by Ni-NTA beads, was mixed with Src-bound Sepharose beads, and the binding was examined by immunoblotting (Fig. 7F). AATYK1A was pulled down with Sepharose beads, indicating that AATYK1A associates directly with Src. To assess whether association of AATYK1A with Src requires palmitoylation, association of AATYK1A-C4/6/7S with Src was examined in COS-7 cells by co-immunoprecipitation. The amount of Src co-immunoprecipitated with AATYK1A-C4/6/7S was significantly less than that with AATYK1A (Fig. 7G); the amount of Src bound to AATYK1A-C4/6/7S was approximately 50% of that bound to AATYK1A. These data indicate that palmitoylation of AATYK1A increases the association between AATYK1A and Src.

We verified the co-localization of Src with AATYK1A in COS-7 cells. COS-7 cells expressing AATYK1A and Src were incubated with Alexa-transferrin for 60 min to visualize recycling endosomes. Localization of AATYK1A and Src was observed by immunostaining with anti-AATYK1 and anti-Src, respectively (Fig. 7H, a–d). Higher magnification views of the perinuclear region are shown in Fig. 7G, e–h. Although both AATYK1A and Src showed a punctate staining pattern in the cytosol, the staining patterns differed (Fig. 7H, e–f,I–j). This may be due to localization of the majority of Src in late endosomes (Sandilands et al. 2004). However, it was also apparent from the merged images that some Src-positive puncta coincided with AATYK1A-positive recycling endosomes in the perinuclear region. Co-localization is indicated by white color in the merged images (Fig. 7H, h). These results indicate that a portion of Src associates with AATYK1A in recycling endosomes.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
AATYK1 is a unique protein kinase because, although it is predicted to be a tyrosine kinase based on amino acid sequence of its kinase domain, tyrosine kinase activity has not been shown experimentally. Further, neither the function nor the cellular localization of AATYK1 has been clearly demonstrated and AATYK1A has only been suggested to be a palmitoylated protein. In this study, we demonstrate that AATYK1A is palmitoylated at cysteine residues 4, 6 and 7 in COS-7 cells and neurons and that palmitoylation targets a portion of AATYK1A to transferrin-positive recycling endosomes. The SFKs, Src and Fyn phosphorylated AATYK1A at tyrosine residues 25 and 46 in a palmitoylation-dependent manner. We further identify Src as a novel AATYK1A-interacting protein; a fraction of Src co-localized with AATYK1A in transferrin-positive recycling endosomes and co-immunoprecipitated with AATYK1 from an endosome-enriched brain microsomal fraction. These data indicate that palmitoylation of AATYK1A plays critical roles in its endosomal localization and association with Src.

AATYKs are protein kinases on the cytoplasmic surface of membranes. AATYK2, AATYK3 and AATYK1B are integral membrane proteins with an N-terminal transmembrane sequence (Manning et al. 2002; Wang & Brautigan 2002; Kawa et al. 2004; Tomomura et al. 2007). AATYK1A does not contain the transmembrane sequence but associates with membranes via palmitoylation. Considering the potential reversibility of palmitoylation, AATYK1A may reversibly bind to membranes and thereby function in a manner distinct from other AATYKs. In this study, we could not distinguish between AATYK1A and AATYK1B. Thus, clarification of localization and functional differences between these isoforms awaits development of their specific antibodies.

AATYK1A seems to localize to membrane compartments differently than the other AATYK family proteins. Although most of AATYK2 and AATYK3 fractionates with detergent-resistant membrane microdomains (Tomomura et al. 2007), AATYK1 in mouse brains largely fractionates with detergent-soluble membranes (Tomomura et al. 2007 and our unpublished data). The majority of AATYK1 in detergent-soluble fractions appears to correspond to AATYK1A because AATYK1A expressed in COS-7 cells was obtained in the detergent-soluble fraction after sucrose density gradient centrifugation (our unpublished data). In contrast, AATYK1B showed a different cellular distribution from AATYK1A. AATYK1B displayed a reticular staining resembling endoplasmic reticulum when expressed in COS-7 cells (Supporting Information Fig. S3), as was reported in cultured neurons (Tomomura et al. 2007). A small fraction of endogenous AATYK1 was obtained in the detergent-resistant membrane fraction as AATYK2 and AATYK3; it is hypothesized that this fraction contains mainly AATYK1B. Palmitoylation is also found in integral membrane proteins, such as ion channels and G-protein-coupled receptors, as is the case for peripheral membrane proteins (Papac et al. 1992; Hayashi et al. 2005). AATYK1B, AATYK2 and AATYK3 also have cysteine residues in the cytoplasmic region close to the transmembrane domain, the region corresponding to palmitoylation sites of AATYK1A, suggesting that they may also be palmitoylated. If so, palmitoylation of these AATYKs would have another function, such as targeting to detergent-resistant membrane microdomains, than the membrane association we showed here with AATYK1A.

AATYK proteins appear to reside mainly in intracellular membrane organelles. We observed that AATYK1A localized as punctate staining in the perinuclear region of COS-7 cells, as also observed in human neuroblastoma SHSY5Y cells (Raghunath et al. 2000). We found that a portion of AATYK1A accumulated in perinuclear recycling endosomes in COS-7 cells. Perinuclear AATYK1A partially localized with a trans-Golgi marker, consistent with the fact that recycling endosomes are located in a perinuclear region opposing the trans-Golgi network with some intercommunication. AATYK1A exhibited a punctate distribution in neurites as well as the perinuclear region in cultured neurons, in agreement with localization of recycling endosomes in neurons (Prekeris et al. 1999). Recycling endosomes are involved in a number of transport pathways, including receptor recycling to the plasma membrane. In neurons, for example, recycling endosomes supply AMPA receptors to the postsynaptic region (Park et al. 2004) and are required for dendritic spine growth (Park et al. 2006). AATYK1A might also be involved in such synaptic function in the postsynaptic region by regulating trafficking of endosomes. Recently, it was shown that AATYK2 (LMTK2), which also localizes to endosomes in transfected HeLa cells, interacts with myosin VI, an actin-based motor protein (Chibalina et al. 2007). Although the interaction of AATYK1A with motor proteins has not been reported, AATYK1A might be involved in regulation of endosomal trafficking.

AATYKs were originally suspected to be tyrosine kinases based on sequence comparison with receptor tyrosine kinases. Subsequent reports described a serine/threonine kinase activity for AATYKs (Wang & Brautigan 2002, 2006; Kawa et al. 2004). We detected phosphorylation of AATYK1A tyrosines catalyzed by Fyn or Src and an association with Src. We identified the phosphorylation sites as tyrosines 25 and 46. The amino acid sequence downstream of tyrosine 46 constitutes a minimal consensus sequence, YXXP (YVLP in AATYK1A), for interaction with the SH2 domain of various signaling proteins, including, for example, Abl, phospholipase C, Crk and Nck (Songyang et al. 1993). AATYKs have also been shown to bind other protein kinases and protein phosphatases. For example, Cdk5/p35 binds and phosphorylates AATYK1 and AATYK2 at serine/threonine residues (Honma et al. 2003; Kesavapany et al. 2003). Further, AATYK1A associates with SPAK/PASK and protein phosphatase 1 (Gagnon et al. 2007). Thus, AATYK1 may provide a platform on endosomes where several protein kinases or protein phosphatases assemble.

Src localizes to endosomes, particularly late endosomes (Kaplan et al. 1992), although its role in endosome function remains unclear. Src translocates from RhoB-positive late endosomes to the plasma membrane when Src is activated in fibroblasts by platelet-derived growth factor stimulation (Sandilands et al. 2004, 2007). This translocation of Src is blocked when the recycling pathway is inhibited by expression of mutant Rab11, a small GTPase that regulates vesicle recycling, suggesting that Src passes through recycling endosomes when it translocates to peripheral membranes. Src may transiently associate with AATYK1A during transit from late endosomes to the plasma membrane. Even if this is the case, Src could interact with and phosphorylate AATYK1A residing in recycling endosomes, possibly to form the signaling platform required for endosomal trafficking. It would be useful to search for proteins that interact with AATYK1 dependent on tyrosine phosphorylation to show unknown functions of AATYK1A.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Chemicals and antibodies

Chemicals and antibodies used in this study included PP2, PP3 and anti-Src (clone 327) (Calbiochem, La Jolla, CA), leupeptin (Peptide Institute, Osaka, Japan), 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and anti-synaptotagmin (Wako Pure Chemical Industries, Osaka, Japan), monoclonal (9E10) and polyclonal anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA), anti-PSD-95 (Affinity BioReagents, Golden, CO), anti-β-tubulin, monoclonal (M2) and polyclonal anti-FLAG, and 2-bromopalmitate (Sigma, St. Louis, MO), anti-NR2A (Chemicon, Temecula, CA), anti-transferrin receptor (Zymed Laboratories, South San Francisco, CA), mouse anti-GM130 and anti-dynamin (BD Transduction Laboratories, San Diego, CA), anti-Golgin-97 and Alexa-conjugated secondary antibodies (488, 546 and 647) (Invitrogen Corp., Carlsbad, CA), and anti-phosphotyrosine (4G10) (Upstate Biotechnology, Lake Placid, NY). Anti-AATYK1 was produced by immunizing rabbits with a GST-AATYK1A fragment composed of amino acids 651–853 (M651-853) and was affinity-purified using a column of Hi-Trap NHS-activated Sepharose (GE Healthcare, Piscataway, NJ) conjugated with recombinant His-tagged AATYK1A-M651-853. Pervanadate (10 mM) was freshly prepared by mixing Na3VO4 (20 mM) and H2O2 (20 mM) for 10 min at room temperature.

Plasmid construction

Full-length mouse AATYK1A-FLAG and AATYK1B-FLAG cDNA were constructed in the pCAGGS expression vector as described by Tomomura et al. (2001, 2007). To construct AATYK1A-Myc-His, AATYK1A was amplified by PCR using pCAGGS-AATYK1A-FLAG as a template and then cloned into pcDNA3.1-Myc-His (Invitrogen). AATYK1A deletion mutants, corresponding to amino acid residues 1–1130 (N1130), 1–840 (N840), 1–667 (N667), 1–390 (N390), 78–667 (M78-667) and 1–667 without 78–351 ({Delta}KD) were constructed by PCR using AATYK1A-Myc-His as a template. Schematic representations of AATYK1A deletion mutants used in this study are shown in Supporting Information Fig. S2A. The GST-AATYK1A constructs containing residues 1–78 (N78) or 651-853 (M651-853) were inserted into pGEX 4T-1 (GE Healthcare) after PCR amplification using AATYK1A cDNA as a template. AATYK1A with mutations at putative palmitoylation sites (C4/6/7A, C4S, C6S, C7S, C4/6S, C4/7S, C6/7S and C4/6/7S) or tyrosine phosphorylation sites (Y25F, Y46F and Y25/46F) was constructed using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. The nucleotide sequence of all constructs was confirmed by sequencing. pME-Fyn (Fyn, Fyn-Y531F and Fyn-K109M), pME-FLAG-Fyn-Y531F, and pcDNA3-Src (Src and Src-Y530F) were kindly provided by T. Tezuka and T. Yamamoto at the University of Tokyo.

Cell culture, transfection and preparation of cell extracts

COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum, 100 U/mL penicillin and 0.1 mg/mL streptomycin. Transfection into COS-7 cells was carried out using the Polyfect transfection reagent (Qiagen, Hilden, Germany) or Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cerebral cortical neurons were prepared from ICR mice embryonic day 15–16 (SLC, Tokyo, Japan) as described (Wei et al. 2005). Cortical neurons cultured for 6 days (6 DIV) were transfected using the Calcium Phosphate Profection kit (Promega, Madison, WI). For immunostaining analysis, cortical neurons were maintained in Neurobasal medium supplemented with B-27 supplement and 0.5 mM L-glutamine on poly-D-lysine-coated coverslips. In some experiments, we treated neurons with 2-bromopalmitate to inhibit palmitoylation. The treatment with 100 µM 2-bromopalmitate for up to 8 h did not cause any harmful effect on neurons.

Cells were disrupted by passing through a 27-gauge needle 10 times in 20 mM HEPES pH 7.5, 2 mM MgCl2, 1 mM EGTA, 0.4 mM AEBSF and 10 µg/mL leupeptin. After centrifugation at 1000 g for 5 min, the soluble and insoluble fractions were obtained by centrifugation of the supernatant at 100 000 g for 1 h. The membranes-containing pelleted material was suspended in RIPA buffer (20 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 0.15 M NaCl, 1% Nonidet P-40, 0.1% SDS, 0.1% sodium deoxycholate, 10 mM β-glycerophosphate, 10 mM NaF, 1 mM Na3VO4, 0.4 mM AEBSF, 10 µg/mL leupeptin) to make the volume equal to that before centrifugation.

Cultured neurons or mouse brains were homogenized in 4 mM HEPES pH 7.5, 0.32 M sucrose, 1 mM EGTA, 0.4 mM AEBSF and 10 µg/mL leupeptin. The homogenates were centrifuged at 800 g for 10 min to obtain the postnuclear supernatant (S1). S1 was centrifuged at 10 000 g for 15 min to separate the synaptosomal fraction as the pellet (P2) and soluble or microsomal components as the supernatant (S2). After osmotic shock–induced disruption of the synaptosomes in P2, the PSD-enriched (LP1) and presynaptic membrane–enriched (LP2) fractions were obtained in the pellets after sequential centrifugations at 25 000 g for 20 min and at 540 000 g for 2 h, respectively (Huttner et al. 1983). High-density microsome (HDM) and low-density microsome (LDM) fractions were obtained from the S2 fraction by centrifugation at 25 000 g and 540 000 g, respectively, for 2 h. Protein concentrations were determined with the Coomassie protein assay reagent (Pierce Biotechnology Inc., Rockford, IL) using bovine serum albumin as a standard.

Immunofluorescence staining of cultured cells

COS-7 cells or cultured neurons were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, and permeabilized with 0.1% Triton X-100 containing 5% normal goat serum. Cells were probed with primary antibodies for 1 h at room temperature or overnight at 4 °C. After washing with PBS, cells were stained with Alexa-conjugated secondary antibody. Fluorescent images were acquired by a Fluoview FV1000 confocal microscope (Olympus, Tokyo, Japan) or a LSM5 EXCITER (Carl Zeiss, Oberkochen, Germany).

Metabolic radiolabeling and immunoprecipitation

Cortical neurons or COS-7 cells expressing AATYK1A proteins were cultured in the presence of 1 mCi/mL [3H]palmitate (Moravek Biochemicals, Brea, CA) for 4–8 h (Hayashi et al. 2005). The labeled cells were washed with ice-cold PBS and lysed in RIPA buffer. The supernatant produced after centrifugation at 10 000 g at 4 °C for 20 min was subjected to immunoprecipitation with anti-Myc (9E10) or anti-AATYK1 antibody using protein G-Sepharose (GE Healthcare). Immunoprecipitated proteins were detected by immunoblotting after SDS-polyacrylamide gel electrophoresis (PAGE). Palmitoylation was detected by autoradiography using a Bas2000 imaging analyzer (FujiFilm, Tokyo, Japan).

Phosphorylation of AATYK1A

Fyn tyrosine kinase was prepared from COS-7 cells expressing FLAG-Fyn by immunoprecipitation with anti-FLAG (M2). GST-AATYK1A-N78 was incubated with Fyn in kinase buffer (10 mM MOPS, pH 6.8, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA) in the presence of 0.1 mM [{gamma}-32P]ATP for 30 min at 35 °C. Phosphorylation was detected by autoradiography after SDS-PAGE. Tyrosine phosphorylation in COS-7 cells was assessed by immunoblotting with anti-phosphotyrosine (4G10) after immunoprecipitation.


   Acknowledgements
 
We would like to express our thanks to T. Tezuka and T. Yamamoto at the University of Tokyo for providing Fyn and Src cDNA, and H. Yakura and K. Mizuno at Tokyo Metropolitan Institute for Neuroscience for use of a confocal microscope. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (S.H.).


   Footnotes
 
Communicated by: Takeo Kishimoto

* Correspondence: tutumi-kouji{at}ed.tmu.ac.jp or hisanaga-shinichi{at}tmu.ac.jp


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Received: 19 May 2008
Accepted: 16 June 2008





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