Protein Interaction Network of Human Protein Kinase D2 Revealed by Chemical Cross-Linking/Mass Spectrometry

We investigated the interaction network
of human PKD2 in the cytosol
and in Golgi-enriched subcellular protein fractions by an affinity
enrichment strategy combined with chemical cross-linking/mass spectrometry
(MS). Analysis of the subproteomes revealed the presence of distinct
proteins in the cytosolic and Golgi fractions. The covalent fixation
of transient or weak interactors by chemical cross-linking allowed
capturing interaction partners that might otherwise disappear during
conventional pull-down experiments. In total, 31 interaction partners
were identified for PKD2, including glycogen synthase kinase-3 beta
(GSK3B), 14–3–3 protein gamma (YWHAG), and the alpha
isoform of 55 kDa regulatory subunit B of protein phosphatase 2A (PPP2R2A).
Remarkably, the entire seven-subunit Arp2/3 complex (ARPC1B, ARPC2,
ARPC3, ARPC4, ARPC5, ACTR3, ACTR2) as well as ARPC1A and ARPC5L, which
are putative substitutes of ARPC1B and ARPC5, were identified. We
provide evidence of a direct protein–protein interaction between
PKD2 and Arp2/3. Our findings will pave the way for further structural
and functional studies of PKD2 complexes, especially the PKD2/Arp2/3
interaction, to elucidate the role of PKD2 for transport processes
at the trans-Golgi network. Data are available via
ProteomeXchange with identifiers PXD003909 (enrichment from cytosolic
fractions), PXD003913 (enrichment from Golgi fractions), and PXD003917
(subcellular fractionation).