PEAK3/C19orf35 pseudokinase, a new NFK3 kinase family member, inhibits CrkII through dimerization - PubMed (original) (raw)

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

Mitchell L Lopez et al. Proc Natl Acad Sci U S A. 2019.

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.

Keywords: CrkII; NKF3 family; motility; protein kinase; pseudokinase.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Identification of PEAK3 (C19orf35) as a homolog of PEAK1 and Pragmin. (A) Protein sequence alignment for the kinase domains of human PEAK1/SgK269, Pragmin/SgK223, and PEAK3/C19orf35 and their corresponding secondary structure elements, which are denoted based on the Pragmin structure (PDB ID code 5VE6). Shading for conserved residues (black), conservative mutations (gray), and canonical sequence motifs of active kinases (red) are marked. Residues involved in dimerization, conserved W and C residues, and those shown to occlude the pseudoactive site in Pragmin are marked by the indicated symbols. (B) Phylogenetic tree (PhyML) for selected NKF3 kinases. Branches with bootstrap values better than 70% are marked with circles; an asterisk (*) represents predicted proteins. (C) Cartoon representation of the crystal structure of the Pragmin SHED domain/pseudokinase module (PDB ID code 5VE6). (D) Zoomed-in view of the pseudoactive site in Pragmin depicting residues that occlude the canonical nucleotide-binding pocket. Top numbering is for Pragmin residues (PDB ID code 5VE6), bottom in parentheses for predicted corresponding residues in PEAK3. The Pragmin D184 residue was modeled in the active site based on the other structure of Pragmin (PDB ID code 6EWX).

Fig. 2.

PEAK3 binds CrkII. (A) Schematic representation of PEAK1, Pragmin, and PEAK3 domain structure. The locations of the CrkII-binding sites and helical regions within the SHED domain are highlighted. (B) Consensus sequence of CrkII-binding sites in selected proteins. (C) Sequence logo depicting conservation of the CrkII-binding site in PEAK3 homologs. (D and E) Coimmunoprecipitation of FLAG-tagged wild-type PEAK3 or a CrkII-binding mutant (PEAK3-3A) transiently expressed in HEK293 cells with either endogenous CrkII (D) or transiently expressed untagged CrkII variants carrying mutations in the SH3 domains (W170K in SH3N and W276K in SH3C) (E). Protein levels were detected with the indicated antibodies by Western blot. All coimmunoprecipitation data are representative of at least 3 independent experiments.

Fig. 3.

PEAK3 prevents CrkII-dependent membrane ruffling and lamellipodia-like extensions. (A) Confocal microscopy imaging of COS-7 cells transiently cotransfected with a FLAG-tagged wild-type or a CrkII-binding deficient mutant of PEAK3 (PEAK3-3A) with either an empty vector or untagged CrkII. CrkII was detected with anti-CrkII antibody (green), PEAK3 with anti-FLAG antibody (blue) and F-actin with Alexa Fluor 647-conjugated phalloidin (red). (Scale bars, 20 μm.) (B) Relative percentage of different actin fiber phenotypes measured in COS-7 cells transiently transfected with PEAK3 and stained with Alexa Fluor 647-conjugated phalloidin (n = 60 cells per group). Cells were blindly scored and binned based on the extent of visible actin fibers within the cytosol: (i) prominent fibers that traversed over 50% of the cell, (ii) short stochastic fibers, and (iii) no significant amount of polymerized actin. (C) Schematic illustrating perimeter calculation in representative cells from A. (D) Average perimeter of COS-7 cells expressing either wild-type or a CrkII-binding deficient (PEAK3-3A) variants of PEAK3 with an empty vector or untagged CrkII, quantified as described in Materials and Methods. Data represent the mean ± SEM of 3 independent experiments (n = 20 cells in each experiment), ***P < 0.001.

Fig. 4.

CrkII-binding motif is insufficient for PEAK3-dependent inhibition of CrkII. (A) Schematic representation of used PEAK3 constructs. (B) Average perimeter of COS-7 cells transiently expressing either wild-type or the ΔPK variants of PEAK3 with an empty vector or untagged CrkII. Data represent the mean ± SEM of 3 independent experiments (n = 20 cells in each experiment), ***P < 0.001. (C) Coimmunoprecipitation of FLAG-tagged wild-type and ΔPK PEAK3 variants, transiently expressed in HEK293 cells, with exogenously expressed, untagged CrkII. Protein levels were detected with the indicated antibodies by Western blot. Data are representative of 3 independent experiments.

Fig. 5.

PEAK3 dimerization via the pseudokinase/SHED module is necessary for binding and inhibition of CrkII. (A) Coimmunoprecipitation of a FLAG-tagged and an HA-tagged variant of wild-type PEAK3, transiently expressed in HEK293 cells. Protein levels were detected with the indicated antibodies by Western blot. (B, Upper) Pragmin SHED/pseudokinase domain (PK) dimer structure (PDB ID code 5VE6). (Lower) Dimerization interface and interactions between helices within the SHED domain. Residues colored in magenta were selected for mutagenesis in PEAK3 based on sequence homology. Top numbering corresponds to Pragmin (PDB ID code 5VE6), bottom in parentheses to PEAK3. (C_–_F) Coimmunoprecipitation of dimerization-deficient variants of PEAK3 with wild-type PEAK3 (C and D) or with untagged CrkII (E and F), all transiently expressed in HEK293 cells. Proteins levels were detected with the indicated antibodies by Western blot. All coimmunoprecipitation data are representative of 3 independent experiments. (G and H) Average perimeter of COS-7 cells expressing wild-type or dimerization-deficient variants of PEAK3 with an empty vector or untagged CrkII. Data represent the mean ± SEM of 3 independent experiments (n = 20 cells in each experiment), ***P < 0.001.

Fig. 6.

Role of PEAK3 pseudokinase domain in regulation of CrkII extends beyond dimerization. (A) Schematic representation of used PEAK3 constructs. (B) Coimmunoprecipitation of untagged CrkII with FLAG-tagged dimerization-deficient mutants of PEAK3, transiently expressed in HEK293 cells. Protein levels were detected with the indicated antibodies by Western blot. (C) Average perimeter of COS-7 cells transiently expressing either wild-type or diCC variant of PEAK3 with an empty vector or untagged CrkII. Data represent the mean ± SEM of 3 independent experiments (n = 20 cells in each experiment), ***P < 0.001. (D and E) Coimmunoprecipitation of the PEAK3 D330N variant with wild-type PEAK3 (D) or untagged CrkII (E) transiently expressed in HEK293 cells. Proteins levels were detected with the indicated antibodies by Western blot. All coimmunoprecipitation data are representative of 3 independent experiments. (F) Average perimeter of COS-7 cells transiently expressing either wild-type or the D330N variant of PEAK3 with an empty vector or untagged CrkII. Data represent the mean ± SEM of 3 independent experiments (n = 20 cells in each experiment), *P < 0.05, ***P < 0.001.

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PEAK3/C19orf35 pseudokinase, a new NFK3 kinase family member, inhibits CrkII through dimerization - PubMed (original) (raw)