// Miao Zhao 1 , Matthias Spiess 1 , Henrik J. Johansson 2 , Helene Olofsson 1 , Jianjiang Hu 1 , Janne Lehti&ouml; 2 and Staffan Str&ouml;mblad 1 1 Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden 2 Cancer Proteomics Mass Spectrometry, Department of Oncology-Pathology, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden Correspondence to: Staffan Str&ouml;mblad, email: Staffan.Stromblad@ki.se Keywords: p21-activated kinase 4, actin cytoskeleton, protein-protein interaction, mass spectrometry, VCA domain Received: May 13, 2017&emsp;&emsp;&emsp;&emsp; Accepted: July 25, 2017&emsp;&emsp;&emsp;&emsp; Published: August 18, 2017 ABSTRACT p21-activated kinase 4 (PAK4) regulates cell proliferation, apoptosis, cell motility and F-actin remodeling, but the PAK4 interactome has not been systematically analyzed. Here, we comprehensively characterized the human PAK4 interactome by iTRAQ quantitative mass spectrometry of PAK4-immunoprecipitations. Consistent with its multiple reported functions, the PAK4 interactome was enriched in diverse protein networks, including the 14-3-3, proteasome, replication fork, CCT and Arp2/3 complexes. Because PAK4 co-immunoprecipitated most subunits of the Arp2/3 complex, we hypothesized that PAK4 may play a role in Arp2/3 dependent actin regulation. Indeed, we found that PAK4 interacts with and phosphorylates the nucleation promoting factor N-WASP at Ser484/Ser485 and promotes Arp2/3-dependent actin polymerization in vitro. Also, PAK4 ablation in vivo reduced N-WASP Ser484/Ser485 phosphorylation and altered the cellular balance between G- and F-actin as well as the actin organization. By presenting the PAK4 interactome, we here provide a powerful resource for further investigations and as proof of principle, we also indicate a novel mechanism by which PAK4 regulates actin cytoskeleton remodeling.

Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization