Role of DHHC3 tyrosine phosphorylation in the control of its expression and functional activity

S-palmitoylation is a postranslational addition of palmitate, a 16-carbon saturated fatty acid to cysteine residues through a labile thioester linkage, which is catalyzed by protein acyl transferases (PAT). Recently the zinc finger DHHC (Asp-His-His-Cys) type-containing protein family has emerged as a large family of palmitoyl acyltransferases with 23 members in the mouse and human genomes. Among them DHHC3 (also known as Golgi-specific DHHC zinc finger protein, GODZ) was shown as a relatively promiscuous palmitoyltransferase, which palmitoylate a broad range of substrates. These substrates includes signaling proteins (as several G protein α subunits), synaptic vesicle proteins (as SNAP-25 and cysteine-signaling protein), scaffold proteins (PSD-95), ion channels (γ2 subunit of GABAA receptor; GluR1 and 2) and cell adhesion molecules (as α6 and β4 integrin subunits and NCAM). Palmitoylation of neurospecific substrates of DHHC3 was shown to be important for synaptic function, plasticity, neuronal migration and maturation of neurons. Considering this, lack or gain of DHHC3 function could be a key point in progression of neurodegenerative diseases. So the exploration of regulation of DHHC3 activity is of the particular interest. Tyrosine phosphorylation is one of the most common regulatory posttranslational modifications and turns many protein enzymes on and off, thereby altering their function and activity. DHHC3 has several tyrosines which are potential sites for src and FGFR mediated phosphorylation. First we have shown that WT DHHC3 cotransfected in N2a cells with src or FGFR1 became highly tyrosine phosphorylated. Treatment of the cells with PP2 – a selective src inhibitor - reduces the phosphorylation of DHHC3, which is even more decreased if PP2 is applied together with FGFR inhibitor PD 173074. The DHHC3 Y-F mutant in which all 5 tyrosines (Y) at the cytoplasmic domains were mutated to phenylalanines (F) shows tyrosine phosphorylation neither under basal conditions nor in response to src or FGFR1 overexpression. Generating single and triple Y-F mutants (two sites 295, 297 on the C-terminus of the protein were mutated together) we were able to dissect the contribution of each site to the basal level of tyrosine phosphorylation and also to src or FGFR1 mediated hyperphosphorylation. Two tyrosines 295, 297 at the C-teminus of DHHC3 are responsible for activation directly by src-mediated pathway, while tyrosine located at the N-terminus of the protein in the position 18 is important for phosphorylation in response to signaling downstream of FGFR1. Interestingly, tyrosines 295, 297 contribute probably also to stability of DHHC3, since mutants lacking this site are expressed at significantly lower level than WT. It appeared that DHHC3tyrosine phosphorylation interferes with its autopalmitoylation: the full DHHC3Y-F mutant is 2 times more palmitoylated than the WT. The autopalmitoylation gives insight into understanding the catalytically function of the enzyme, because in accordance with the two-step ping-pong theory autopalmitoylation is an intermediate step in transferring the palmitate to the protein substrate. Hence, our ongoing study aims to verify if a lack of tyrosine phosphorylation of DHHC3 would affect its activity towards its substrates and play a role in the neuronal development and plasticity.