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. Author manuscript; available in PMC: 2019 Aug 9.
Published in final edited form as: Bioquim Patol Clin. 1998;62(1):5–17.

Purification and molecular characterization of NP185, a neuronal-specific and synapse-enriched clathrin assembly polypeptide.

Shengwen Li 1, Michael Lisanti 2, Saul Puszkin 3
PMCID: PMC6688760  NIHMSID: NIHMS1038344  PMID: 31402847

Abstract

NP185, a neuronal-specific protein of 185 kDa, was first discovered when we prepared monoclonal anti-bodies (mAbs) against bovine brain clathrin coated vesicles. Two mAbs, 8G8 and 6G7, permitted us to characterize this protein both biochemically and in development (NP185 is expressed in a NGF-dependent manner in PC12 cells). The expression of NP185 coincides with synaptogenesis. In this work, we have further characterized this protein as follows: Microsequence analysis of immuno-purified native NP185 from bovine brain yielded five peptides that corresponded exactly to the known sequences of murine F1–20 and rat AP180 (renamed AP3); ii) Using an established assay, we show that purified recombinant NP185/AP3 can facilitate clathrin cages assembly; iii) Using deletion mutagenesis, we mapped the epitopes of two distinct mAbs directed against bovine NP185 to a 60 amino acid residue region of the murine recombinant NP185/AP3; iv) Recombinant NP185/AP3 can be phosphorylated by purified casein kinase II in vitro; and v) Recombinant NP185/AP3 directly binds to purified brain tubulin. Since NP185/AP3 binds to tubulin and stimulates the clathrin assembly, it may be involved in the regulation of the transport of clathrin-coated vesicles. Casein kinase II, an enzyme known to be present in clathrin-coated vesicles, may play a role in the regulation of NP185/AP3 for the promotion of clathrin assembly.

Keywords: NP185/AP3, phosphorylation, casein kinase II, tubulin binding, epitope mapping, microsequencing

Introduction

Clathrin coated vesicles (CCVs) participate in various intracellular transport processes, such as receptor mediated endocytosis and the retrieval of membrane after exocytosis. Biochemical and morphological evidence shows that clathrin-coated vesicles play a major role in the nervous system—specifically in the recycling of synaptic vesicle membranes after they fuse and discharge their contents within the synaptic cleft (Maycox et al., 1992). Neuronal-specific components of clathrin coated vesicles have been identified. These include neuronal specific isoforms of the clathrin light chains (LCa and LCb) (Jackson et al., 1987; Kirchhausen et al., 1987b) and neuronal specific isoforms of members of the assembly polypeptides, AP-2 complex (Robinson, 1990; Robinson, 1989) that facilitates the assembly and attachment of clathrin to plasma membranes.

NP185 (Neuronal Protein of 185 kDa) is a major component of clathrin coated vesicles (Kohtz and Puszkin, 1988; Puszkin et al., 1992). This 185 kDa protein was first identified when we elicited mAbs against clathrin coated vesicles purified from bovine brain (Kohtz and Puszkin, 1988). Two mAbs (8G8, 6G7), which specifically recognized NP185, have allowed us to study the distribution and localization of NP 185 (Kohtz and Puszkin, 1988; Kohtz and Puszkin, 1989; Perry et al., 1991; Perry et al., 1992; Su et al., 1991). Unlike clathrin—the major coat protein of coated vesicles, - the expression of NP185 is highly restricted. It is only expressed in brain tissue and selectively in neurons (Kohtz and Puszkin, 1988; Kohtz and Puszkin, 1989; Perry et al., 1991; Perry et al., 1992; Su et al., 1991). In PC12 cells, a model for neuronal cell - systems, NP185 is dramatically induced by stimulation with nerve growth factor (NGF) during differention (Kohtz and Puszkin, 1988). These developmental studies showed that expression of NP185 coincided with synaptogenesis (Perry et al., 1991; Puszkin et al., 1992). This molecule is enriched in mature synaptic terminals, associated with clathrin light chains and casein kinase II (Kohtz and Puszkin, 1989; Su et al., 1991).

However, little is known about the function of NP185 in brain cellular activities. Clues to the function of NP185 came from in vitro reassembly experiments using purified clathrin triskelia (Su et al., 1991). In these experiments, NP185 acted as an assembly promoter agent that facilitated the assembly of clathrin into cages under conditions where clathrin was unable to assemble on its own. NP185 behaved like other assembly polypeptides—multiple-subunit complexes termed AP-1 and AP-2 (AP, assembly polypeptide)— that were expressed in most if not all cell types (Keen, 1990; Robinson, 1992).

Ungewickell and colleagues have identified a 180 kDa protein that they termed AP180 (Ahle and Ungewickell, 1986), while Keen and colleagues discovered pp155 (Keen and Black, 1986). In many respects, NP185, AP180 and pp155 share similar properties (Ahle and Ungewickell, 1986; Keen and Black, 1986; Kohtz and Puszkin, 1988), and they were therefore renamed as AP3 (Murphy et al., 1991). However, this is based solely on immunological cross-reactivity. It was very difficult to purify a sufficient amount of the native NP185 for microsequencing analysis since the native NP185 is extremely sensitive to proteolysis. Thus, there was no direct evidence to confirm the identity from the amino acid sequence of purified native NP185.

Here, we provide direct molecular evidence for the identity of NP185 with AP180/F1–20 (designated as AP3). Immuno-purification and microsequencing of native bovine NP185 yielded five peptide sequences that were identical to the known sequence of rat AP-180 and murine F1–20 that is a protein identified by immunoscreening of cDNA expression library. Using the murine F1–20/AP3 cDNA we specifically mapped the epitopes of two mAbs directed against NP185 to a 60 amino acid residue region of NP185/AP3. Also, we demonstrate that bacterial-expressed recombinant murine NP185/AP3 functionally promoted the assembly of clathrin triskelia into cages. Further experiments showed that recombinant NP185/AP3 directly interacts with brain tubulin and is phosphorylated by casein kinase II (CKII) in vitro.

Experimental Procedures

Immuno-affinity purification of NP185.

Partial purified native NP185 was isolated from bovine brain clathrin coated vesicles (CCVs) (Schook and Puszkin, 1985) by high salt extraction (Kohtz and Puszkin, 1988). Extracted proteins were separated on Sepharose CL6B and subsequently on Mono Q ion-exchange chromatography. Fractions containing native NP185 were passed through antibody affinity column (See Figure 1A). Pure native bovine NP185 was eluted from this column, and separated by SDS-PAGE. The protein was visualized by staining with Coomassie blue.

Figure 1. Immuno-affinity purification of native NP185 from extracts of bovine brain coated vesicles.

Figure 1.

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A) Purification scheme. Native bovine NP185 was successfully purified by differential centrifugation, sizing columns (gel filtration columns), ion exchange column (Mono Q column), and immuno-affinity chromatography. Anti-NP185 mAb (8G8) was coupled to CNBr-activated Sepharose 4B for the immuno-affinity chromatography.

B) Immuno-affinity chromatography with anti-NP185 mAb indicates that there are two isoforms of native NP185 from bovine brain. Peak 1: minor NP185 peak, in which the NP185 Isoform has low affinity to the anti-NP185 mAb therefore eluted first from the column; Peak 2: major NP185 peak, which was used for further analysis and microsequencing, in which the NP185 isoform has high affinity to the NP185 mAb therefore eluted second from the column.

C) Purified native bovine NP185. Left, Coomassie Blue staining; Right, Immunoblot analysis with anti-NP185 mAb 8G8. Lane 1, purified clathrin coated vesicles; lane 2, soluble extract of clathrin coated vesicles; lane 3, purified native bovine NP185. Note abbreviations: HC, clathrin heavy chain; LC, clathrin light chains. Note that the purified native bovine NP185 does not react with an anti-clathrin heavy chain antibody and anti-assembly polypeptide antibody, indicating the purity of the native bovine NP185 (not shown).

Microsequence Analysis.

The pure native bovine NP185 protein in gels was excised and subjected to microsequencing at The Protein Chemistry Group, Biotechnology Resource Laboratory, W.M. Keck Foundation (by Dr. Kathy Stone, Yale University). Internal peptides were obtained by trypsin digestion of the purified native bovine NP185. The enzymatic cleavage fragments were separated on a narrow-pore (2.1-mm i.d.) reverse-phase high-performance liquid chromatography (HPLC) using a dual-syringe Brownlee micropump (Aebersold et al., 1987). The peptides were sequenced by automated gas-phase sequenator. The amino acid sequences of those peptides were analyzed using the programs FASTA and BLAST through the GenBank database.

Immunoblotting

Mouse brain homogenates were prepared as described previously (Sousa et al., 1992). Native NP185 obtained from the high salt extract of bovine brain CCVs (Kohtz and Puszkin, 1988) as well as affinity purified protein were used for immunoblotting analysis. Gel electrophoresis was adapted for the brain preparations following the procedure of Laemmli (1970). Sample preparations were loaded onto 5–15% gradient SDS-PAGE gels. Proteins were then transferred to nitrocellulose filters for immunoblotting analysis with mouse anti-NP185 mAbs (Kohtz and Puszkin, 1988) or anti-F1–20 mAbs (kindly provided by Dr. E. M. Lafer) (Sousa et al., 1990). Goat anti-mouse secondary antibody IgG conjugated with alkaline phosphatase. After washings, the signals were detected by BCIP/NBT in developing buffer.

Reverse Transcriptase-PCR cloning of the murine F1–20 cDNA.

Mouse brain poly (A+) RNA (1 μg) was used for reverse transcription with the first-strand cDNA synthesis kit (Pharmacia) by following the manufacturer’s instructions. The first-strand cDNA sample (15 μl) was heated to 90°C for 5 minutes to denature the RNA-cDNA duplex and to inactivate the reverse transcriptase, then chilled on ice. The entire first-strand cDNA sample was then amplified in PCR using primers flanking the open reading frame of F1–20 cDNA, forward: 5’-CTCACTCGAGGGCCGGT-GAAGATGTC-3’ and reverse: 5’-CTCAACTCGA-GAATCTTATCTGAAGTTTCC-3.’ (Zhou et al., 1992; Zhou et al., 1993).

The purified PCR product of the expected size was end-repaired and ligated with Smal cut pGEX-3X to generate the recombinant plasmid that expresses GST-NP185/AP3 fusion protein. The recombinant plasmids with correct orientation and exact reading frame were determined by restriction enzyme mapping and double-stranded DNA sequencing. The GST-NP185/AP3 fusion protein was obtained when expressed in a suitable Escherichia coli strain (BL21, lacking Lon and ompT proteinases, Novagen, Inc.).

Purification of GST-N185/AP3 fusion protein.

Purification of GST-NP185/AP3 protein was performed using glutathione Sepharose 4B column (Pharmacia) according to manufacturer’s instruction with following modification (Li et al., 1995; Li et al., 1996a; Marston, 1986). Briefly, the induced cells were harvested by low speed centrifugation. Pellet was washed by the buffer of 25 mM Tris-HCI, pH 7.5, 10 mM EDTA, 85 mM NaCI and 50 mM glucose. Proteinase inhibitors were immediately added to the cells and the cells were frozen in liquid nitrogen. After thawing, the sample was treated by Polytron and sonicated and centrifuged. The supernatant was diluted in PBS (150 mM NaCI, 16 mM Na2HP04, 4 mM NaH2P04, pH 7.3), plus 1% Triton X-100, and applied to the glutathione-agarose affinity column that specifically binds to GST-portion of fusion protein. After washing for eight times, the fusion protein was eluted with reduced glutathione-containing buffer. All manipulations were performed at 4°C (in cold room) or on ice, and all solutions were ice-cold and contained the following mixture of protease Inhibitors: phenylmethylsulfonyl fluoride (1 mM), aprotinin (2 mg/ml), pepstatin (1 mg/ml), and leupeptin (1 mg/ml).

Clathrin assembly assay.

An established assay was used to evaluate the effect of native bovine NP185 and recombinant NP185/AP3 on clathrin assembly into cages (Su et al., 1991). Clathrin triskelions were purified as described previously following the procedure of Winkler and Stanley (Winkler and Stanley, 1983). The effect of NP185 on the polymerization of clathrin was determined by layering purified clathrins incubated with either purified native bovine NP185 or the purified GST-NP185/AP3 fusion protein, on top of a 10% sucrose solution. The samples were centrifuged at 100,000g to permit polymerized structures to sediment at the bottom of the sucrose layer. Aliquots from the top solution and the resuspended pellets were assayed by either immunoblotting or ELISA to detect clathrins and NP185. The pellets were resuspended for electron microscopy to visualize the type of structure formed.

Approximately 10 μg of the pellet containing NP185-induced clathrin cage polymers were suspended in 0.1 M Tris buffer pH 7.0 and used for negative staining. Samples were placed on Formvar-coated grids, negatively stained with 1 % uranyl acetate, air dried and examined in a Joel 100-B electron microscope at an acceleration voltage of 80 kV (Su et al. 1991).

Epitope-mapping of mAbs 8G8 and 6G7.

For expression of NP185/AP3 as a recombinant GST-fusion protein, the cDNA for murine F1–20/AP3 was subcloned in frame into the multiple cloning site of the vector pGEX3X or pGEX-1λT. Deletion mutants were then created by partial digestion with Pst I or by PCR using primers that incorporated convenient restriction sites. Fusion proteins were then expressed in E.coli strain BL21 after IPTG induction, purified and used as the substrate for immunoblotting with mAbs 8G8 and 6G7 directed against native NP185.

In Vitro phosphorylation

In vitro phosphorylation was performed as described previously (Li et al., 1996b). Recombinantly expressed purified Casein Kinase II (UBI, Inc.) was incubated with GST alone, GST-FL-NP185/AP3 (Full-length NP185/AP3 fused to GST) bound to glutathione Sepharose beads. After 2 hour incubation with rotation at 4°C, GST or GST-NP185/AP3 bound to beads were washed extensively with a buffer containing SDS and deoxycholate (Li et al., 1996b; Pendergast et al., 1991; Pendergast et al., 1991). The NP185-CKII complex or GST alone bound to the beads was then incubated with kinase reaction buffer containing 20 mM Hepes, 5 mM MgCI2, 1 mM MnCI2, γ−32P-ATP, for 5–10 minutes at room temperature. The reaction was stopped by adding the SDS sample buffer and boiling at 100 oC for 5 min. After SDS-PAGE, dried gel was subjected to autoradiography to visualize phosphorylated proteins.

Interaction of brain tubulin and recombinant NP185/AP3

We have adopted an established assay to determine the interaction of brain tubulin and recombinant NP185/AP3 (Li et al., 1995). Brain tubulin was purified by DEAE chromatography (gift from Dr. David R. Colman, Columbia University). Approximately 5 μg of either GST or purified GST-NP185/AP3 fusion proteins bound to glutathione-agarose beads were extensively washed with phosphate-buffered saline (PBS) (six times) and incubated with 2 μg of brain tubulin in 1 ml of PBS containing proteinase inhibitors by rotating overnight in the cold room (4°C). After binding, the beads were extensively washed (for six times) with PBS containing proteinase inhibitors. Finally, bound proteins were eluted with 100 ml of elution buffer containing PBS, 10 mM reduced glutathione and proteinase inhibitors. The eluate was mixed 1:1 with 2 X SDS-sample buffer and separated on a 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting with anti-tubulin (1:1000 dilution) monoclonal antibody. Horse radish peroxidase-conjugated secondary antibodies (1:5000 dilution; Amersham Corp.) were used to visualize bound primary antibodies by enhanced chemiluminescence (ECL).

Results

Immuno-purification and microsequencing analysis of native bovine NP185: Identity with murine F1–20 phosphoprotein and rat AP180.

Native bovine NP185 is a large molecule (185 kDa) and extremely sensitive to proteinase degradation. It is masked by dominant 180 kDa of clathrin heavy chain and very acidic so that it was escaped from detection with any conventional techniques such as SDS-PAGE and Coomassie blue staining. Native NP185 tightly associates with other molecules such as assembly polypeptides of 100 kDa and 50 kDa. Thus, it was difficult to purify native NP185 as a single species and in sufficient amounts for protein microsequencing. Therefore, uncovering of the molecular structure of the protein awaited purification from recommbinant 185 expressed molecules.

Here, we have developed a procedure to successfully purify native bovine NP185 to be homogeneity through differential centrifugation, different buffer extraction, gel filtration chromatography, ion exchange chromatography, immuno-affinity chromatography, and HPLC (outlined in Figure 1A). Through the immuno-affinity chromatography with anti-NP185 mAb, native bovine NP185 was eluted in two peaks: a minor peak (Peak 1) and a major peak (Peak 2) (Figure 1B). These two peaks may represent two NP185 isoforms (Kohtz and Puszkin, 1988). The fraction of the peak 1 represents an isoform of NP185 that has a low binding affinity for it’s antibody and it was eluted first from the column. The fraction of the peak 2 represented an isoform with high affinity with the anti-NP185 mAb. After immuno-affinity chromatography, the purified protein appeared as a single band by SDS-PAGE analysis and Coomassie Blue staining (Figure 1C, lane 3). The fraction from the major peak (Peak 2) was used for further analysis. Purified native NP185 did not cross-react with antibodies directed against other coated vesicle components such as clathrin or the assembly polypeptides (not shown).

We then performed microsequencing analysis of immuno-purified native bovine NP185. As the N-terminus was blocked (Li and Puszkin, 1991, unpublished observations), NP185 was digested with trypsin and internal peptides were isolated by HPLC (15.5 mq protein: ~ 100 picomoles, approximately from 15 freshly obtained bovine brains; See Experimental Procedures). The five internal peptide sequences obtained are listed in Table I. These five peptides did not show homology to any known genes in the GenBank in 1992 except that they corresponded exactly to the known sequence of murine F1–20 phosphoprotein and rat AP180, which were deposited into the GenBank but not yet published. These results clearly demonstrate that at the primary amino acid sequence level, native bovine NP185 and AP3 (murine F1–20/rat AP180) are apparently identical which may be due to extremely similar genes. In support of this assumption, immuno-purified native bovine NP185 reacted with a monoclonal antibody probe raised against murine F1–20 (Figure 2).

Figure 2.

Figure 2.

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Recognition of purified native bovine NP185 by anti-NP185 and anti-F1–20 mAbs.

Purified native bovine NP185 (isolated as described in Figure 1) was resolved by SDS-PAGE and electroblotted onto nitrocellulose membranes. Strips were cut and probed with anti-NP185 mAbs (8G8 and 6G7) and anti-F1–20 mAb. The arrow marks the position of purified full-length native bovine NP-185. Note that some minor degradation products are observed, as native NP185 is extremely sensitive to proteolysis.

Murine F1–20 is a neuronal phosphoprotein of unknown function identified by immunoscreening of cDNA expression library (Zhou et al., 1992); rat AP180 Is a 180 kDa clathrin assembly polypeptide (Ahle and Ungewickell, 1986). Rat AP180 and murine F1–20 are now believed to represent the rat and the murine forms of the same gene product (Morris et al., 1993; Zhou et al., 1993). Keen and colleagues have used the term AP3 for the Isoforms of bovine pp155, rat AP180, bovine NP185 (Murphy et al., 1991) and murine F1–20 (Morris et al., 1993; Zhou et al, 1993).

Recombinant expression and assembly promoting activity of purified recombinant NP185/AP3.

We isolated the cDNA for murine F1–20 by reverse transcriptase-polymerase chain reaction (RT-PCR) from mouse brain mRNA. The sequence of PCR product was confirmed by restriction mapping and DNA sequencing. The entire coding region was subcloned into the vector pGEX-3X for recombinant expression as a GST-fusion protein. Immunoblot analysis revealed that the recombinant NP185/AP3 fusion protein is specifically recognized by an anti-NP185 mAb (8G8) and an anti-F1–20 mAb (Figure 3). These results independently support our results from microsequencing analysis of immuno-purified native bovine NP185.

Figure 3. Recombinant expression of full-length F1–20/NP185(AP3) as a GST fusion protein.

Figure 3.

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Total proteins from the E. coli cells containing the recombinant plasmid encoding recombinant F1–20/NP185(AP3) were resolved by SDS-PAGE. A plus “+” indicates that IPTG was used to induce expression of the F1–20 fusion protein. Left, Coomassie Blue staining; Middle, Immunoblot analysis with anti-NP185 mAb 8G8; Right, Immunoblot analysis with anti-F1–20 mAb (F1–20). Note that recombinant F1–20/NP185(AP3) is specifically recognized by both anti-NP185 (8G8) and anti-F1–20 monoclonal antibodies. Full-length GST-F1–20/NP185(AP3) migrated with an apparent molecular weight of ~ 210 kDa and is indicated by arrow.

Clathrin triskelia can self-assemble into regular coat structures—clathrin cages in vitro under certain conditions (e.g., pH 6.5) even in the absence of membranes. It is, however, strictly dependent on protein cofactors, namely assembly polypeptides under certain conditions (e.g., pH 7.5). Using an established in vitro assembly assay, we have shown that native bovine NP185 can induce clathrin to assemble under conditions that do not favor clathrin assembly (Su et al., 1991). In this sense, native NP185 behaves similarly to other assembly polypeptide complexes, namely AP-1 and AP-2 (Keen, 1990).

To investigate whether recombinant NP185/AP3 can function as a facilitator of clathrin assembly, we purified the full-length recombinant fusion protein GST-NP185/AP3 by affinity chromatography on glutathione agarose and gel-elution (Figure 4A). Like native bovine NP185, recombinant NP185/AP3 promoted the assembly of purified clathrin triskelia into clathrin cages. As observed with other assembly polypeptides, the addition of recombinant NP185/AP3 induced the formation of clathrin cages of a relatively uniform diameter, 98% of the cages with 60–80 nm in diameter (Figure 4 B). As a control, GST failed to induce the formation of clathrin cages (Figure 4 C). Additional controls, recombinant NP185 alone or clathrin alone) did not show any clathrin cage structures under the same conditions (not shown). Further SDS-PAGE and immunoblotting analysis show that the reassembled cages contained intact recombinant NP185/AP3 and clathrins (not shown).

Figure 4. Purified recombinant NP185/AP3 fusion protein promotes the assembly of clathrin triskelions into cages.

Figure 4.

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The GST-FL-NP185/AP3 fusion protein was first purified by affinity chromatography on glutathione agarose and then resolved by SDS-PAGE. In order to obtain the full-length protein product, the band corresponding to the full-length GST-F1–20/ NP185(AP3) fusion protein (as indicated by immunoblot using anti-NP185 mAb) was excised from the gel and eluted according to the manufacturer’s instructions (Amicon). After gel-elution, the full-length fusion protein was used to assay for clathrin assembly activity.

A) Lane 1, Total proteins from cells containing the recombinant plasmid induced by IPTG was resolved by SDS-PAGE and stained by Commassie blue; lane 2, GST-F1–20/ NP185 (AP3) fusion protein purified by affinity chromatography on glutathione agarose and gel-elution.

B) Purified GST-F1–20/ NP185 (AP3) fusion protein promotes the assembly of clathrin triskelions into cages. Clathrin was incubated with the purified full-length fusion proteins in Tris buffer at pH 7.5, and the protein mixture was layered on top of a 10% sucrose solution and centrifuged at high speed in an airfuge for 2 hours. The pellet formed under the sucrose layer was resuspended and an aliquot deposited on a Fromvar-coated grid. Scanning electron microscopy shows that the formation of clathrin cages are induced by the bacterially expressed GST-F1–20/ NP185/AP3 fusion protein. X 120,000. Note that clathrin triskelions can not assemble into cages at pH 7.5 without the presence of assembly polypeptides. The white bar indicates 100 nm.

C) Purified GST alone did not stimulate clathrin triskelions to form cages under the same condition as the recombinant NP185/AP3. The white bar indicates 100 nm.

Epitope mapping of anti-bovine-NP185 mAbs (8G8 and 6G7).

We mapped the epitopes of mAbs directed against bovine NP185 by deletion mutagenesis of the murine NP185/AP3 cDNA (Figure 5). Deletion mutants were generated by complete and partial digestion with Pstl; smaller fragments of NP185/AP3 were generated by PCR amplification using selected primers as described in Experimental Procedures. The epitopes for both mAbs directed against bovine NP185 (8G8 and 6G7) mapped to the same region of the predicted NP185/AP3 amino acid sequence (411–471) as shown by immunoblot analysis of the deletion mutants (Figure 5; Table II). The position of this epitope relative to other known antibody epitopes is summarized in Figure 6.

Figure 5. Epitope mapping of anti-NP185 mAbs 8G8 and 6G7.

Figure 5.

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A) Schematic diagram summarizing the immuno-reactivity of mAbs 8G8 and 6G7 with a panel of murine NP185/AP3 fusion proteins. The nucleotide positions included within a given insert are as indicated at the endpoints. Convenient restriction sites used in these constructions are also as indicated. Note that both anti-NP185 mAbs 8G8 and 6G7 recognize an epitope within murine NP185/AP3 amino acid residues 411–471 (Corresponding to nucleotide positions 1233–1416).

B) The epitopes for both mAbs 8G8 and 6G7 map to murine NP185/AP3 residues 411–471. Lane 1, total proteins from E.coli BL21 cells expressing GST fusion protein encoding amino acid residues 411–471 of murine NP185/AP3 (Corresponding to nucleotide positions 1233–1416); lane 2, total proteins of cells expressing GST alone; lane 3, total proteins of cells expressing full-length GST-F1–20/ NP185 (AP3). Left panel, immunoblot analysis with anti-NP185 mAb 8G8; right panel, immunoblot analysis with another anti-NP185 mAb 6G7.

Figure 6.

Figure 6.

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Schematic diagram summarizing the epitopes of anti-NP185, anti-F1–20, and anti-AP180 monoclonal antibodies.

In vitro phosphorylation of NP185/AP3 by purified casein kinase II.

AP3 was previously Identified as phosphoprotein in vivo by several laboratories. Puszkin and colleagues found that native bovine NP185 associates with casein kinase II activity (CKII) (Kohtz and Puszkin, 1989; Su et al., 1991). Ungewickell and colleagues have demonstrated that AP180 is phosphorylated in serine residues in clathrin-coated vesicles from bovine brain (Ahle and Ungewickell, 1986; Morris et al., 1990). Similarly, with metabolic labeling, Keen and colleagues show that pp155 is phosphorylated in serine residues (Keen and Black, 1986; Murphy et al., 1994). Lafer’s group suggested that F1–20 is a phosphoprotein using phosphatase treatment and phosphatase inhibitors (Zhou et al., 1992). Taken together, NP185/AP3 is phosphorylated presumably by serine kinases. Indeed, cDNA sequence analysis revealed that there are seven consensus motifs for casein kinase II in murine F1–20 (Zhou et al., 1992). However, there is no evidence to support that AP3 can be directly phosphorylated by casein kinase II.

In this work, we studied the serine phosphorylation of NP185/AP3 by casein kinase II in vitro. We reconstituted that serine phosphorylation of NP185/AP3 by purified recombinant casein kinase II in vitro. Recombinant full-length NP185/AP3, expressed as a GST-fusion protein, was incubated with purified recombinant casein kinase II in the presence of [g-32P]ATP. Under these conditions, only NP185 underwent casein kinase ll-mediated phosphorylation. GST alone failed to be phosphorylated by CKII, despite the fact that it contains serine residues. Omission of either casein kinase II or [γ−32P]ATP prevented phosphorylation, indicating that this phosphorylation is ATP- and CKII-dependent (Figure 7). Recombinant NP185/AP3 appeared not to be phosphorylated by either purified recombinant Src tyrosine kinase or purified recombinant Fyn-tyrosine kinases (not shown).

Figure 7. In vitro phosphorylation of recombinant full-length murine NP185/AP3 by casein kinase II.

Figure 7.

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Purified full-length NP185/AP3 (GST-FL-NP185) expressed as a GST fusion protein or GST alone was subjected to in vitro phosphorylation with purified recombinant casein kinase II and γ−32P-ATP in kinase reaction buffer. After SDS-PAGE, phosphorylated proteins were visualized by autoradiography. Note that each reaction contained equivalent amounts of GST and purified full-length recombinant NP185/AP3, although only the recombinant NP185/AP3 undergoes phosphorylation. Phosphorylation of purified full-length recombinant NP185/AP3 was specifically dependent on addition of both casein kinase II and γ−32P-ATP. Omission of either casein kinase II and γ−32P-ATP prevented phosphorylation.

Interaction of NP185 with brain tubulin

Previous findings suggested that native bovine NP185 is associated with brain tubulin(Kohtz and Puszkin, 1989; Su et al., 1991). Here, we used an established assay to determine the potential direct Interaction between brain tubulin and recombinant NP185/AP3 (Li et al., 1995). We expressed full-length NP185/AP3 (GST-FL-NP185 as indicated in Figure 8) as a GST fusion protein. After purification by affinity chromatography on glutathione-agarose, GST-NP185/AP3 fusion protein bound on the beads were incubated with purified brain tubulin. After extensive washings, GST fusion proteins bound to glutathione-agarose beads were specifically eluted with reduced glutathione-containing buffer. As this treatment releases GST fusion proteins, it also indirectly releases proteins bound to GST fusion proteins; non-specifically bound proteins should remain attached to the beads under these conditions. To evaluate brain tubulin binding, these eluates were then subjected to SDS-PAGE analysis and immunoblotting with anti-tubulin monoclonal antibody. As compared with a critical control GST alone, brain tubulin bound specifically to full-length NP185/AP3, but not to GST alone (Figure 8). This is the first indication that NP185/AP3 can directly bind to brain tubulin.

Figure 8.

Figure 8.

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Interaction of brain tubulin and recombinant NP185/AP3. GST-FL-NP185/AP3 (Bound to glutathione-agarose beads) was incubated with brain tubulin purified by DEAE column chromatography (gift from Dr. David R. Colman). GST-FL-NP185/AP3 represents full-length recombinant NP185/AP3 (residues 1–902) expressed as a GST fusion protein. After binding, extensive washing (six times), and eluted with reduced glutathione, bound tubulin was visualized by Western blotting with a tubulin-specific monoclonal antibody. As a critical control, binding was compared with GST alone. In addition, note that antibody binding was specifically dependent on the addition of tubulin, demonstrating that the antibody does not cross-react with either recombinant NP185/AP3 or GST. Equivalent amounts of GST and GST-FL-NP185/AP3 (about 1 ug per binding experiment) were used in binding experiments. For each binding experiment, 0.2 ug of native brain tubulin was used which was purified by DEAE chromatography.

Discussion

Among the principal results of this study was the purification and molecular characterization of NP185, a synapse-specific protein. We learned that the microsequence analysis of immuno-purified native bovine NP185 yielded five peptides that correspond exactly to the known sequences of murine F1–20 and rat AP180, which was recently designated as AP3; also using an established assay, we showed that purified recombinant NP185/AP3 can act as a facilitator of clathrin assembly. With deletion mutagenesis, we mapped the epitopes of two distinct mAbs directed against NP185 to a 60 amino acid residue region of murine NP185/AP3; we detected that NP185/AP3 is phosphorylated by casein kinase II in vitro and that NP185/AP3 directly binds to brain tubulin.

Bovine NP185 has been discovered and characterized biochemically in our lab (Kohtz and Puszkin, 1988; Kohtz and Puszkin, 1989; Su et al., 1991), and immuno-histochemically in mouse and chicken (Perry et al., 1991; Perry et al., 1992; Puszkin et al., 1992); while murine F1–20 has been cloned by immunoscreening of cDNA expression library without any evidence showing a relationship with other proteins (Zhou et al., 1992). Indeed, a survey of previously published reports indicates that NP185, pp155, AP180 and F1–20 have a number of striking similarities: i) expression only in neuronal cells (Kohtz and Puszkin, 1988; Sousa et al., 1992); ii) synapse- specific (Perry et al., 1991; Sousa et al., 1992) ; iii) all are phosphoproteins (Keen and Black, 1986; Morris et al., 1989; Morris et al., 1990; Zhou et al., 1992); iv) all are acidic and with anomalous migration on SDS- PAGE (Ahle and Ungewickell, 1986; Keen and Black, 1986; Kohtz and Puszkin, 1988; Zhou et al., 1992); v) all are developmentally regulated in their expression (Perry et al., 1991; Sousa et al., 1992); and vi) all are different isoforms (Kohtz and Puszkin, 1988; this report; Zhou et al., 1992, 1993; Morris, et al., 1993). However, to date there was no direct evidence for the molecular identity of purified native NP185 with the other forms. Thus, our current results now directly demonstrate that native bovine NP185 shares identity with rat AP180, murine F1–20, bovine pp155. They may represent the different species-specific isoforms of this protein, namely AP3.

One of the conclusions of our work is that NP185/AP3 functions as an adaptin-like molecule since recombinantly-expressed NP185/AP3 induces in vitro clathrin assembly. Previously, we showed that partially purified preparations of native bovine NP185 induce rapid and effective in vitro assembly of clathrin molecules into closed cages (Su et al., 1991); identical results were obtained by others with purified native AP180 (Ahle and Ungewickell, 1986). However, preparations of native NP185 or native AP180 used in those assays may contain assembly polypeptides AP2 contaminants, which, as observed, bind to NP185/AP3, and could be responsible for inducing coat assembly (Kohtz and Puszkin, 1988). To resolve this ambiguity, we obtained recombinantly-expressed and purified NP185/AP3 from bacterial cell lysates. Consequently, we showed that the preparation of recombinant NP185/AP3 is sufficient to induce clathrin cage formation, acting as a facilitator of clathrin-assembly in vitro. Thus, NP185/AP3 is a bona fide assembly polypeptide as defined by this in vitro assay system used to characterize the assembly activity of other more well-established assembly polypeptide complexes—namely AP-1 and AP-2. This confirmed the findings made independently by Lafer and colleagues (Ye and Lafer, 1995).

Two of the monoclonal antibodies directed against native bovine brain NP185, 8G8 and 6G7, have been invaluable for the study of native NP185 structure and function. We have used these antibodies for biochemical characterization of native NP185 (Kohtz and Puszkin, 1988; Kohtz and Puszkin, 1989; Su et al., 1991), examining developmental expression (Perry et al., 1991) , detection of native NP185 in the neuromuscular junction (Perry et al., 1992), as well as in immuno-affinity purification of native NP185 (this report). Since both mAbs can recognize NP185 by immunoblotting after transfer from SDS-PAGE gels, it is reasonable to conclude that the epitopes are sequential, not conformational. Here, we have mapped the epitopes of these mAbs to a discrete 60 amino acid residue region of NP185/AP3 that contains two casein kinase II motifs for potential phosphorylation of NP185/AP3 (Table 2). Therefore, these two anti-NP185 mAbs may be useful for functional characterization of NP185/AP3 phosphorylation sites both in vivo and in vitro. In addition, as summarized in Fig. 6, the epitope of murine F1–20 mAb was found in the middle of F1–20 sequence—between amino acid residue 400 and residue 760 (Zhou et al., 1992) ; while the epitope of rat AP180 mAb was defined to reside between amino acid residue 562 and residue 744 (Morris et al., 1993). These four mAbs could be used as complementary molecular probes to further study the structure and function of AP3. These types of studies may be required when the involvement of these polypetides in diseases of the nervous system is elucidated. The significance of its brain specificity is an indication that those types of studies are overdue.

Native NP185 has been shown to be associated with casein kinase Il (CKII) (Kohtz and Puszkin, 1989; Su et al., 1991). NP185 is threfore involved in membrane transport events associated with neurite development like found in PC12 cells (Kohtz and Puszkin, 1988). Since casein kinase II (CKII) is enriched in the brain and its involvement in neuritogenesis is well established (Ulloa et al., 1993; Wong et al., 1996), the regulated interactions of these two molecules in brain acquires high significance. It’s transient depletion in neuroblastoma cells blocks neuritogenesis indicating CKII regulates neuritogenesis. In neuronal cells, rapid responses to signal transmission take place in the synapse. Specific subcellular localization of CKII might be needed to limit its activity to potential targets involved in regulated rapid synaptic vesicle recycling or fast axonal transport to the synapse. As such, anchoring of CKII to subcellular organelles may sequester the kinase to appropriate substrates and compartmentalize its activity in the synapse. Indeed, the bacterially-expressed NP185/AP3 fusion protein binds to brain tubulin. Thus, NP185/AP3 mediates the interaction of vesicular organelles with cytoskeletal protein, e.g., by binding to tubulin present at the opening base of these organelles. These correlations suggest an increased importance of NP185 in regulating CKII activity for higher neuronal functions. In support of this interpretation, a recent report shows that casein kinase II does play an important role in AP-1 functions for downstream sorting event of theTrans-Golgi vesicular membrane network (Mauxion et al., 1996). In conclusion, NP185 interacts with major functional molecules, structural molecules, and membrane in brain. This role is assigned in nature to molecules who can also be responsible for disease conditions. This exciting possibility awaits further study.

Acknowledgments

We thank the members of Dr. Saul Puszkin’s laboratory for their excellent technical assistance and helpful discussions. We thank Dr. Eileen M. Lafer (University of Pittsburgh) for providing anti-F1–20 mAb and Dr. David Colman (Columbia University) for the purified bovine brain tubulin. This work was supported in part by grants from the United States National Institutes of Health (NIH) NS12467 and NS26113. Dr. Saul Puszkin has been Jacob Javitz awardee by NIH for excellence in Neuroscience. Dr. Michael Lisanti is an awardee of Research Grants by NIH. Dr. Shengwen Li was supported in part by a Research Fellowship award by NIH.

List of Abbreviations:

AP180

assembly polypeptide 180 kDa

CCV

clathrin coated vesicle

CKII

casein kinase II

NGF

nerve growth factor

NP185

neuronal-specific protein 185 kDa

PBS

phosphate-buffered saline

PC12

rat pheochromocytoma cell line

RT-PCR

reverse transcriptase polymerase chain reaction

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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