Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation

Abstract

The pseudo-kinase and signaling protein Pragmin has been linked to cancer by regulating protein tyrosine phosphorylation via unknown mechanisms. Here we present the crystal structure of the Pragmin 906-1368 amino acids C-terminus, which encompasses its kinase domain. We show that Pragmin contains a classical protein kinase fold devoid of catalytic activity. A particular inhibitory triad, conserved in a Pragmin/SgK269/PEAK1/C19orf35 superfamily, tightly holds the catalytic lysine (K997) to prevent ATP binding. By proteomics, we discovered that this pseudo-kinase uses the tyrosine kinase CSK to induce protein tyrosine phosphorylation in human cells. Interestingly, the protein kinase domain is bordered by N-and C-terminal extensions forming an original dimerization domain that regulates Pragmin self-association and stimulates CSK activity. A1329E mutation in the C-terminal extension destabilizes Pragmin dimerization and reduces CSK activation. Thus, our results reveal a new dimerization mechanism by which a pseudo-kinase can induce protein tyrosine phosphorylation.All rights reserved. No reuse allowed without permission.

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

“…Likely as a consequence of these changes, both kinases were demonstrated to lack the ability to bind nucleotides (4). Consistent with these data, the recent crystal structures of both PEAK1 and Pragmin revealed nucleotide-binding pockets with significantly distorted architectures, which render them incompatible with ATP binding (3,5,6). Collectively, these observations support the categorization of PEAK1 and Pragmin as pseudokinases.…”

“…S1). In the PEAK1 and Pragmin crystal structures, these conserved motifs are involved in the interactions between the pseudokinase domain and the SHED domain (3,5,6). PEAK3 has remarkably well-conserved sequences in these regions, indicating that the SHED domain is likely present in PEAK3 adopting a similar structure to the ones in PEAK1 and Pragmin (SI Ap-pendix, Fig.…”

“…In PEAK1 and Pragmin, the catalytic lysine is also present but is "hijacked" by interactions with 3 residues collectively termed the "inhibitory triad" that, in addition to other conserved residues, occlude the nucleotide binding pocket and prevent binding of ATP ( Fig. 1 C and D) (3,5,6). These residues are well-conserved in PEAK3 and are represented by D184, Y187, L201, Q231, and L311 ( Fig.…”

“…Predicted SHED Domain in PEAK3. Recent crystal structures of the PEAK1 and Pragmin C-terminal domains have revealed identical dimer forms composed of the pseudokinase domains interacting through a set of helical bundles, termed the SHED domain (3,5,6,12). The SHED domain is unique to NKF3 proteins and is comprised of the helix immediately N-terminal to the pseudokinase domain (αN helix) and 3 helices C-terminal to the pseudokinase domain (αJ, αK, αL helices) that form an "XL"-shaped helical bundle [lettering of helices reflect Pragmin nomenclature (6); PEAK1 helices are off-set by one letter from the αJ helix (5)] (3, 5, 6).…”

“…C19orf 35, which was only recently annotated as PEAK3, is a new human kinase-like protein in the New Kinase Family 3 (NKF3) that contains 2 known pseudokinases: pseudopodium enriched atypical kinase 1 (PEAK1, also called Sugen kinase [SgK] 269) and Pragmin (PEAK2/SgK223) (1,3). PEAK1 and Pragmin are large (1,746 and 1,406 residues, respectively) signaling scaffolds comprised of long N-terminal regions with no predicted domain structure followed by protein kinase-like domains at their C termini.…”