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As illustrated in Figure 4.2, VAMP4 contains two potential CK2 phosphorylation sites in its N-terminal domain, one serine residue at position 20, and one at position 30. In order to determine if either or both of these residues is the phosphorylation site, I performed site directed mutagenesis and changed these serine residues to alanine. As shown in Figure 5.2 A, both VAMP4 L25, 26V and VAMP4 S20A are phosphorylated with comparable efficiency to the wildtype protein. Phosphorylation is dependent on the presence of CK2, excluding that VAMP4 undergoes autophosphorylation. However, VAMP4 S30A is no longer phosphorylated. Accordingly, the double mutant, where both potential phosphorylation sites are mutated, VAMP4 S20,30A, is not phosphorylated in vitro, either. These results demonstrate that, under these conditions, serine 30 is the only CK2-phosphorylation site of VAMP4. Similarly, the protein is no longer phosphorylated if serine 30 is mutated to aspartate. (Figure 5.2B).

Phosphorylation o f VAM P4 modulates its interaction with AP-1 162 Mw [kDal 45 — 31 — wt + wt L25V_{L26V S20A} S20A S30A S30A -H - f - -t- + GST- VAMP4 CK2 B -k - 4- - 4- - -H CK2 VAMP4 V A M P 4 V A M P 4 p o ^ wt S30A S30D Mw [kDal 6 7 - 4 5 - Coomassie 31 - 21.5 — 6 7 - 4 5 - Autoradiography 31 - 21.5 - CK2

Phosphorylation of VA M P4 m odulates its interaction with AP-1____________________ 163

Figure 5.2 Serine 30 is the CK2-phosphorylation site o f VAMP4. In vitro kinase assay o f wt and mutant VAMP4 GST-fusion proteins. Equal amounts o f GST-fusion proteins were incubated with CK2 and P]GTP and subjected to SDS-PAGE and autoradiography. A) Phosphorylation is dependent on the presence of CK2,

excluding that the protein undergoes autophosphorylation. GST-VAMP4 L25, 26V and GST-VAMP4 S20A are phosphorylated with efficiencies comparable to the wt protein. GST-VAMP4 S30A and the double mutant GST-VAMP4 S20,30A can not he phosphorylated, suggesting that serine 30 is the phosphorylation site o f VAMP4. B) Neither GST-VAMP4 S30A nor GST-VAMP4 S30D can he phosphorylated, confirming that serine 30 is the phosphorylation site o f VAMP4. The Coomassie stained gel is shown to confirm equal protein load.

Phosphorylation of V A M P4 m odulates its interaction w ith AP-1____________________ 164

Because the pre-treatment of VAMP4 with GTP and CK2 resulted in improved binding of AP-1, it was hypothesised that the S30A mutant should still bind to AP-1, however, this interaction should not be influenced by kinase treatment. In addition, a mutant VAMP4 protein was produced where serine 30 was m utated to aspartate in order to mimic the phosphorylated state by adding negative charge to the protein. If the aspartate mutant would indeed mimic the phosphorylated state, and if phosphorylation would indeed modulate the interaction of VAMP4 and AP-1, then one would expect improved interaction between the proteins that would be insensitive to kinase treatment.

Accordingly, a GST-pulldown assay with wt GST-VAMP4, GST-VAMP4 S30A, and GST-VAMP4 S30D was performed (Figure 5.3). As shown before (Figure 4.6 and 4.7), wt GST-VAM P4 shows improved binding to AP-1 if it is phosphorylated prior to incubation with bovine adrenal medulla cytosol. VAMP4 S30A interacts with AP-1 with a similar efficiency to the non-phosphorylated wt VAMP4 fusion protein. This interaction is not enhanced if GST-VAM P4 S30A is pre-treated with CK2, confirm ing the hypothesis that VAMP4 phosphorylation modulates the interaction with AP-1. GST- VAMP4 S30D interacts with AP-1 with comparable efficiency to the phosphorylated wt protein. This interaction is no longer affected by addition of CK2, supporting the hypothesis that VAMP4S30D mimics the phosphorylated state of VAMP4.

In order to investigate if serine 20 is important for the binding of AP-1 at all, GST- pulldowns using VAMP4 mutants where serine 20 is mutated to alanine or aspartate were performed. The result is shown in Figure 5.4. Binding of GST-VAM P4 to AP-1 is abolished if serine 20 is mutated to either alanine or aspartate. However, if VAMP4 is phosphorylated prior to incubation with cytosol, interaction with AP-1 can be detected. This interaction is slightly less efficient compared to the non-phosphorylated wt GST- VA M P4. Thus, in this m utant the interaction w ith AP-1 is rendered strictly phosphorylation dependent. Accordingly, binding is completely abolished with GST- VAMP4 S20,30A, as observed for GST-VAM P4 L25,26A. Serine 20 appears to be crucial for the interaction with AP-1, and even the relative conservative exchanges to aspartate or alanine diminish the interaction between VAMP4 and AP-1 and render it completely phosphorylation dependent.

Phosphorylation of VAMP4 modulates its interaction with AP-1 165

wt

S30A

S30D

Mw

10%

IkDa]

116

GST-

VAMP4

CK2

AP-1

Ponceau

VAMP4

Figure 5.3 Phosphorylation ofVAM P4 modulates the interaction with AP-1. Puli down assay with wt GST-VAMP4, GST-VAMP4 S30A, and GST-VAMP4 S30D and bovine adrenal medulla cytosol. All GST fusion proteins were incubated with ImM GTP, with or without 100 p \} CK2 prior to the incubation with cytosol. Kinase and nucleotides were removed before addition of cytosol. Wt GST-VAMP4 shows binding to AP-1 that is modulated by the presence of CK2. GST-VAMP4 S30A shows binding to AP-1 with comparable efficiency to non-phosphorylated wt protein. Binding is not enhanced by CK2. GST-VAMP4 S30D binds to AP-1 with comparable efficiency to the phosphorylated wt protein, suggesting that the S30D mutant mimics the phosphorylated state of VAMP4. Binding was not further enhanced by CK2. The Ponceau stained nitrocellulose membrane is shown to control for equal load of fusion proteins.

Phosphorylation of VAMP4 modulates its interaction with AP-1 166