Recent mass-spectrometric analysis of the in vivo
phosphorylation state of Densin-180 from tryptic digests of murine PSD preparations revealed a surprising number of phosphorylated serine and threonine residues (Trinidad et al. 2006
; Munton et al. 2007
) (, Table S1
). Except for Ser1392, all of them are located N-terminal of the proposed TMD. Further, analysis of human lung cancer tissue identified two sites of tyrosine phosphorylation within the 826–1213 cluster (http://www.phosphosite.org
; Rikova et al. 2007
). These findings are at odds with the widely accepted topology as shown in , as these phosphorylated residues would reside on the extracellular side of Densin-180.
The experimental evidence supporting extensive phosphorylation in the region of residues 826–1213 is extremely strong. The identification of these phosphorylation sites was accomplished by tandem mass spectrometry using stringent statistical interpretation criteria, combined with manual inspection of the individual spectra. In many cases multiple identifications of the same phosphorylation site was obtained from overlapping tryptic peptides, which were a result of incomplete tryptic digestion. The phosphorylation sites were identified across multiple experiments, and several sites independently identified by multiple laboratories. While a small percentage of phosphorylation sites identified by large-scale mass spectrometry experiments (or any analytical approach for that matter) will turn out to be incorrect, depending on the stringency applied to the interpretation of the tandem mass spectra the arguments listed above effectively address any concerns regarding whether the region from 826 to 1213 is extensively phosphorylated.
This phosphorylation-rich area identified in mouse is highly conserved in rat (99% sequence identity) where all potential sites of phosphorylation are present, and in human (90% sequence identity) with all potential sites present except for S1132.
Potential phosphorylation sites of the C-terminal segment have been investigated in previous studies on recombinant fusion proteins. These experiments identified phospho-Ser1288 and phospho-Ser1392 (all sites renumbered according to murine Q80TE7-1) with a potential role in Densin-180/CamkIIα interaction (Strack et al. 2000
; Walikonis et al. 2001
Mass-spectrometric analysis of native Densin-180 confirmed phospho-Ser1392, but phospho-Ser1288 was not detected (Walikonis et al. 2001
; Trinidad et al. 2006
; Munton et al. 2007
). A failure to identify phospho-Ser1288 to date is possibly due to intrinsic limitations of mass-spectrometric analysis of very complex mixtures of tryptic digests (Kuster et al. 2005
), or due to a low stoichiometry for that site in the particular samples analyzed.
Taken together, the mass-spectrometric results clearly identified multiple phosphorylation sites within the proposed extracellular domain of Densin-180. While there is sparse evidence in the literature for extracellular kinases and a few extracellular phosphorylation sites (e.g., Redegeld et al. 1999
; Canton and Litchfield 2006
), such a clustering of ecto-phosphorylation sites has never been reported and seems highly unlikely. In acknowledgement of these new data we therefore took into consideration the possibility of a different topology, namely an all-intracellular and membrane-associated localization of Densin-180 (), as further outlined below.