PEAK3/C19orf35 pseudokinase, a new NFK3 kinase family member, inhibits CrkII through dimerization

Abstract

Members of the New Kinase Family 3 (NKF3), PEAK1/SgK269 and Pragmin/SgK223 pseudokinases, have emerged as important regulators of cell motility and cancer progression. Here, we demonstrate that C19orf35 (PEAK3), a newly identified member of the NKF3 family, is a kinase-like protein evolutionarily conserved across mammals and birds and a regulator of cell motility. In contrast to its family members, which promote cell elongation when overexpressed in cells, PEAK3 overexpression does not have an elongating effect on cell shape but instead is associated with loss of actin filaments. Through an unbiased search for PEAK3 binding partners, we identified several regulators of cell motility, including the adaptor protein CrkII. We show that by binding to CrkII, PEAK3 prevents the formation of CrkII-dependent membrane ruffling. This function of PEAK3 is reliant upon its dimerization, which is mediated through a split helical dimerization domain conserved among all NKF3 family members. Disruption of the conserved DFG motif in the PEAK3 pseudokinase domain also interferes with its ability to dimerize and subsequently bind CrkII, suggesting that the conformation of the pseudokinase domain might play an important role in PEAK3 signaling. Hence, our data identify PEAK3 as an NKF3 family member with a unique role in cell motility driven by dimerization of its pseudokinase domain.

Probing Protein Interaction Networks by Combining MS-Based Proteomics and Structural Data Integration

“…The first such motif located within the N-terminus of Pragmin and identified by the ELM analysis (Table S6) was the EPIYA sequence, a motif recognized and actively phosphorylated by CSK through its SH2 and protein-kinase domains, respectively. We also detected a proline-rich motif potentially recognized by either PWWP or SH3 domains, and this prediction matches the recently detected binding of CrkII to the equivalent motif present in PEAK3, a recently discovered paralogue of Pragmin 73 . These motifs can explain the detection of Csk and Crk (or its closed homologue CrkL) in the interactome of Pragmin.…”

AXL confers cell migration and invasion by hijacking a PEAK1-regulated focal adhesion protein network

“…PEAK1 contains a putative proline-rich motif that fits the consensus for CRK binding 31 , 32 . To test whether this is involved in their interaction, we generated a mutant of PEAK1 in which this proline-rich region was disrupted (PEAK1 3PA ) and we demonstrated that this nearly eliminated PEAK1 binding to CRKII by both co-IP and glutathione S -transferase (GST) pulldown assays (Fig.…”

PEAK3 pseudokinase represents a pro-migratory and -invasive signalling scaffold

“…The third family member, C19orf35 or PEAK3, was identified by the Roche group in 2018 (Lecointre et al , 2018), although its signalling mechanism and function remain unclear. Recently, it was demonstrated via site-directed mutagenesis and co-immunoprecipitation studies that PEAK3 also homo-dimerizes via the highly conserved SHED domain, this being critical for CrkII recruitment (Lopez et al , 2019). In addition, while PEAK3 overexpression did not impact cell morphology, it prevented CrkII-dependent membrane ruffling, leading the authors to suggest that PEAK3 may represent a negative modulator of PEAK1/2 action (Lopez et al , 2019).…”