Phosphorylation of SNAP-23 by the novel kinase SNAK regulates t-SNARE complex assembly

Abstract

The docking and fusion of cargo-containing vesicles with target membranes of eukaryotic cells is mediated by the interaction of SNARE proteins present on both vesicle and target membranes. In many cases, the target membrane SNARE, or t-SNARE, exists as a complex of syntaxin with a member of the SNAP-25 family of palmitoylated proteins. We have identified a novel human kinase SNAK (SNARE kinase) that specifically phosphorylates the nonneuronal t-SNARE SNAP-23 in vivo. Interestingly, only SNAP-23 that is not assembled into t-SNARE complexes is phosphorylated by SNAK, and phosphorylated SNAP-23 resides exclusively in the cytosol. Coexpression with SNAK significantly enhances the stability of unassembled SNAP-23, and as a consequence, the assembly of newly synthesized SNAP-23 with syntaxin is augmented. These data demonstrate that phosphorylation of SNAP-23 by SNAK enhances the kinetics of t-SNARE assembly in vivo.

Figure 1

Identification of SNAK, a SNARE kinase. (A) Domain structure of SNAK. The presence of the coiled-coil domain within SNAK was identified by the COILS algorithm (Lupas, 1996), and the presence of the kinase domain and the specified subdomains was determined by aligning the primary sequence of SNAK with that of other serine/threonine kinases. (B) Northern blot analysis of SNAK expression. A human tissue Northern blot containing poly(A)⁺ RNA from heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas was probed with a SNAK probe under high-stringency hybridization conditions. A single 4.4-kilobase transcript was detected in all tissues examined in this and other Northern blots.

Figure 2

SNAK phosphorylates SNAP-23 in vitro. (A) GST-SNAK was incubated with immobilized GST, GST-syntaxin 4, or GST-SNAP-23 in the presence of [γ-³²P]ATP at 30°C for 10 min. The reaction was terminated by extensive washing, and in vitro phosphorylation reactions were analyzed by SDS-PAGE and autoradiography. The relative mass (M_r) of ¹⁴C-labeled molecular weight markers is indicated. (B) His₆-SNAP-23 (1.5 μg) was incubated with the indicated amount of GST-SNAK in the presence of [γ-³²P]ATP at 30°C for 30 min. The reaction products were analyzed by SDS-PAGE and autoradiography.

Figure 3

SNAK phosphorylates SNAP-23 in vivo. (A) Transfected HeLa cells expressing syntaxin 4 or SNAP-23 in the absence (−) or presence (+) of SNAK were labeled with [³²P]orthophosphate, lysed in Triton X-100, immunoprecipitated with the indicated antiserum, and analyzed by SDS-PAGE and fluorography. Equivalent fractions of each anti-SNAP-23 immunoprecipitate were also analyzed by immunoblotting with a SNAP-23 antiserum (lower panel). (B) HeLa cells were mock transfected or transfected with 5, 10, or 20 μg of a SNAK expression vector by calcium phosphate coprecipitation and labeled with [³²P]orthophosphate for 2 h. The cells were lysed in Triton X-100, nuclei and debris were removed by centrifugation in a microfuge, and equal amounts of each lysate were analyzed by SDS-PAGE and fluorography. The relative mass (M_r) of ¹⁴C-labeled molecular weight markers is indicated.

Figure 4

SNAP-23 in t-SNARE complexes is not phosphorylated. (A) HeLa cells expressing syntaxin 4, SNAP-23, and SNAK were labeled with [³²P]orthophosphate, lysed in Triton X-100, subjected to immunoprecipitation with the indicated antiserum, and analyzed by SDS-PAGE and fluorography. (B) HeLa cells were transfected with a constant amount of SNAP-23 and SNAK cDNA (2 μg each) and increasing amounts of syntaxin 4 cDNA (0, 0.1, 0.5, or 2 μg) with the use of Lipofectamine. The cells were then labeled with [³²P]orthophosphate, lysed, immunoprecipitated with the indicated antiserum, and directly analyzed by SDS-PAGE and fluorography (upper panel). The immunoprecipitates were also probed by immunoblotting with a SNAP-23 antiserum to detect total SNAP-23 and t-SNARE–associated SNAP-23 (lower panels).

Figure 5

Phosphorylated SNAP-23 resides in the cytosol. HeLa cells expressing syntaxin 4, SNAP-23, and SNAK were labeled for 3 h with [³²P]orthophosphate. After mechanical cell disruption, the postnuclear supernatant was separated into a membrane fraction and a cytosol fraction as described in the text. Triton X-100 was added to each fraction, immunoprecipitations were performed with the use of the indicated antiserum, and equivalent portions of the immunoprecipitates from each fraction were analyzed by SDS-PAGE and fluorography (upper panel). The immunoprecipitates were also probed by immunoblotting with a SNAP-23 antiserum to detect the total amount of SNAP-23 present in each sample (lower panel).

Figure 6

Phosphorylation by SNAK promotes SNAP-23 assembly with syntaxin. (A) HeLa cells expressing SNAP-23 and syntaxin 4 in the absence or presence of SNAK were labeled with [³⁵S]methionine for 15 min and chased in complete medium. After incubation for various times at 37°C, the cells were harvested, lysed in Triton X-100, immunoprecipitated with the use of control (C) or anti-syntaxin 4 serum, and analyzed by SDS-PAGE and fluorography. (B) The relative amount of [³⁵S]SNAP-23 coprecipitated with the syntaxin 4 serum at different times of chase in the absence (□) or in the presence (▪) of SNAK was determined by phosphorimager analysis. The amount of [³⁵S]SNAP-23 present is expressed in arbitrary phosphorimager units (AU). Each value represents the results from two independent experiments with SD.

Figure 7

SNAK kinase activity is required to promote SNAP-23 assembly with syntaxin. HeLa cells expressing SNAP-23 and syntaxin 4 in the presence of wild-type SNAK (left panel) or SNAP-23 and syntaxin 4 in the presence of a catalytically inactive SNAK mutant (right panel) were labeled with [³⁵S]methionine for 15 min and chased for various times. After cell lysis in Triton X-100, lysates were immunoprecipitated with the use of control (C) or anti-syntaxin 4 serum and analyzed by SDS-PAGE and fluorography.

Figure 8

SNAK enhances the expression of newly synthesized SNAP-23. (A) HeLa cells expressing SNAP-23 in the absence or presence of SNAK were labeled with [³⁵S]methionine for 15 min and chased in complete medium. After incubation for various times at 37°C, the cells were harvested, lysed in Triton X-100, immunoprecipitated with anti-SNAP-23 serum, and analyzed by SDS-PAGE and fluorography. The mobility of phosphorylated SNAP-23 (asterisk) was determined by analysis of ³²P-labeled SNAP-23 and [³⁵S]methionine-labeled SNAP-23 on the same SDS-PAGE gel. Note that in the presence of SNAK, phospho-SNAP-23 is observed after the pulse radiolabeling with [³⁵S]methionine. (B) The relative amount of [³⁵S]SNAP-23 precipitated with the SNAP-23 serum in the absence (□) or in the presence (▪) of SNAK was determined by phosphorimager analysis. The recovery of [³⁵S]SNAP-23 for each data point was expressed as a fraction of the amount of SNAP-23 recovered from cells coexpressing SNAP-23 and SNAK after the pulse radiolabeling (time 0). Each value represents the results from two independent experiments with SD.