PSKH1 kinase activity is differentially modulated via allosteric binding of Ca2+ sensor proteins

Abstract

Protein Serine Kinase H1 (PSKH1) was recently identified as a crucial factor in kidney development and is overexpressed in prostate, lung, and kidney cancers. However, little is known about PSKH1 regulatory mechanisms, leading to its classification as a "dark" kinase. Here, we used biochemistry and mass spectrometry to define PSKH1's consensus substrate motif, protein interactors, and how interactors, including Ca²⁺ sensor proteins, promote or suppress activity. Intriguingly, despite the absence of a canonical Calmodulin binding motif, Ca²⁺-Calmodulin activated PSKH1 while, in contrast, the ER-resident Ca²⁺ sensor of the Cab45, Reticulocalbin, Erc55, Calumenin (CREC) family, Reticulocalbin-3, suppressed PSKH1 catalytic activity. In addition to antagonistic regulation of the PSKH1 kinase domain by Ca²⁺ sensing proteins, we identified UNC119B as a protein interactor that activates PSKH1 via direct engagement of the kinase domain. Our findings identify complementary allosteric mechanisms by which regulatory proteins tune PSKH1's catalytic activity and raise the possibility that different Ca²⁺ sensors may act more broadly to tune kinase activities by detecting and decoding extremes of intracellular Ca²⁺ concentrations.

Keywords: UNC119B; allostery; calmodulin; protein kinase; reticulocalbin.

Fig. 1.

PSKH1 is a serine kinase that favors basic residues at the -3 position. (A) Radiometric assay of recombinant full-length PSKH1 expressed in insect cells. Wild-type, but not D218N kinase-dead, recombinant PSKH1 robustly phosphorylates the ADR1 peptide. (B) Schematic of experimental workflow for positional scanning peptide analysis (PSPA) and representative results; schematic created using BioRender. Z denotes fixed positions containing one of the 20 natural amino acids, or either phosphorylated Thr (pT) or phosphorylated Tyr (pY). X denotes variable positions containing randomized mixtures of all natural amino acids except Ser, Thr, and Cys. (C) Radiometric PSKH1 kinase assay performed on peptide array, where darker spots indicate preferred residues. S, G-A-X-X-X-X-X-S-X-X-X-X-A-G-K-K(LC-biotin); T, G-A-X-X-X-X-X-T-X-X-X-X-A-G-K-K(LC-biotin); where X = degenerate mixture of the 16 natural amino acids excluding cysteine, tyrosine, serine, and threonine. All data in panel C and SI Appendix, Fig. S1 are extracted from the same peptide array experiment.

Fig. 2.

Mapping the PSKH1 interactome using complementary proteomic approaches. (A) Schematic of each experimental workflow; created using BioRender. (B) Volcano plot of TurboID proximity labeling experiment in HEL cells with PSKH1 and proximal proteins of interest highlighted. Pink dashed lines denote a threefold change and P-value < 0.05. Data are representative of six independent experiments. (C) Volcano plot of BiCAP, validating interaction of dimerized PSKH1 with UNC119B in HEK293T cells. Pink dashed lines denote a twofold change and P-value < 0.05. Data are representative of four independent experiments. (D) Volcano plot of FLAG-IP, validating interaction of PSKH1 with CREC family members (red dots) and the Cdc37-HSP90 chaperone system (gray). Pink dashed lines denote a fivefold change and P-value <0.05. Data are representative of three independent experiments. In all panels, PSKH1 is highlighted in black, UNC119B in blue, Calcium sensing interactors of the CREC family in red, secretory pathway interactors in purple, and chaperone interactors in gray. Significantly enriched hits are depicted as closed black circles and nonsignificant hits are depicted as open gray circles.

Fig. 3.

Calcium sensor proteins bind and regulate PSKH1 activity. (A) Recombinant PSKH1 interacts with His₆-Calmodulin (CaM) or His₆-RCN3 in a Ca²⁺-dependent manner (Ca²⁺: 500 μM) in Far-western blots, with binding abolished in the presence of the Ca²⁺-chelator, EGTA (10 mM). The interaction was probed using anti-His₆ horseradish peroxidase antibody. Data are representative of two independent replicates. (B) Ca²⁺-CaM promotes, and Ca²⁺-Reticulocalbins (RCN1 or 3) antagonize, kinase activity in a radiometric assay of HA-tagged PSKH1 (10 ng). Ca²⁺-Calumenin (CALU) does not modulate PSKH1 kinase activity. CaM, RCN1, RCN3, and CALU were recombinantly expressed and purified (all 1 μM). Individual data points are shown as circles with accompanying mean ± SD; n = 3. Statistical analysis was performed by one-way ANOVA; *, **, ***, and **** signify P < 0.1, P < 0.01, P < 0.001, and P < 0.0001, respectively; n.s = nonsignificant. (C) Titration of CaM (blue) or RCN3 (orange) (range 0, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1,000 nM) using HA-tagged PSKH1 (10 ng) by radiometric assay. The half-maximal effective concentration (EC₅₀) with CaM is 58.7 nM, while the half-maximal inhibitory concentration (IC₅₀) with RCN3 is 25.7 nM. Data represent mean ± SD; n = 3. (D) Plot of highest and lowest PSKH1 kinase activity from CaM or RCN3 titration. Individual data points are shown as circles with accompanying mean ± SD; n = 3. Statistical analysis was performed by the t test; ** and **** signify P < 0.01 and P < 0.0001, respectively. (E) Titration of CaCl₂ (0, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, and 1,000 μM) with CaM (1 μM; blue) or RCN3 (1 μM; orange) using HA-tagged PSKH1 (10 ng) by radiometric assay. The EC₅₀ of PSKH1 and CaM by Ca²⁺ is 14.3 μM, while the IC₅₀ of PSKH1 and RCN3 by Ca²⁺ is 183 μM. Data represent mean ± SD; n = 3. (F and G) NHS-Diazirine (SDA; 3.9 Å spacer) chemical crosslinking mass spectrometry of recombinant PSKH1 with Calmodulin (F) and RCN3 (G). PSKH1 kinase domain is highlighted in beige, Calmodulin in Blue, RCN3 in orange, and crosslinks as gray dashes. The position of each EF-hand motif is annotated on Calmodulin and RCN3. (H) AlphaFold model of PSKH1. PSKH1 kinase domain is colored gray and the C-terminal flanking helices in dark gray. Due to very low confidence, the region N-terminal of the kinase domain has been omitted. SDA crosslinks for CaM are colored blue, RCN3 are colored orange and those that overlap are in purple. (I) Domain architecture of PSKH1, where individual mutations are annotated in red. N-terminal truncations are denoted by the number of residues deleted (Δ) and C-terminal truncations by the position of the introduced stop (X). (J) Radiometric assay of HA-tagged PSKH1 WT and various truncation mutants (10 ng) in the absence (white) and presence of 100 μM CaCl₂ and 1 μM CaM (blue). Data represent mean ± SD; n = 3. Statistical analysis was performed by two-way ANOVA; **8 and **** signify P < 0.001 and P < 0.0001, respectively. (K and L) AlphaFold3 models of PSKH1 and CaM (K; colored blue) or RCN3 (L: colored orange). PSKH1 kinase domain is colored gray and the C-terminal flanking helices in dark gray. Due to very low confidence, the region N-terminal of the PSKH1 kinase domain has been omitted. EF-hands of RCN3 are annotated. Predicted aligned error (PAE) plots are shown in SI Appendix, Fig. S4 A and B.

Fig. 4.

UNC119B binds and activates the PSKH1 kinase domain independent of PSKH1 lipidation. (A) Domain architecture of PSKH1, where the myristoyl and palmitoyl groups at positions 2 and 3 are annotated. (B) Radiometric assay of HA-tagged PSKH1 WT, G2A, C3S, and a double G2A/C3S mutant (10 ng) in the absence (white) and presence of UNC119B (1 μM; gray). Individual data points are plotted as circles alongside mean ± SD; n = 3. Statistical analysis was performed by two-way ANOVA; **, ***, and **** signify P < 0.01, P < 0.001, and P < 0.0001, respectively. (C) Radiometric assay of HA-tagged PSKH1 WT with CaM (1 μM; blue) or UNC119B (1 μM; gray) individually and CaM-UNC119B combined (1 μM each; red), in the presence of EGTA (1 mM) or CaCl₂ (100 μM). Data represent mean ± SD; n = 3. Statistical analysis was performed by two-way ANOVA; **** signifies P < 0.0001; n.s = nonsignificant. (D) SDA chemical crosslinking mass spectrometry of PSKH1 and UNC119B. PSKH1 kinase domain is highlighted in beige, UNC119B in dark gray, and crosslinks as gray dashes. (E) AlphaFold model of PSKH1. SDA crosslinks from D are mapped onto the cartoon structure in maroon. The PSKH1 kinase domain is colored gray and the C-terminal flanking helices in dark gray. (F) Radiometric assay of HA-tagged PSKH1 WT and various truncation mutants (10 ng) in the absence (white) and presence of UNC119B (1 μM; gray). N-terminal truncations are denoted by the number of residues deleted (Δ) and C-terminal truncations by the position of the introduced stop (X), as per Fig. 3J. Data represent mean ± SD; n = 3. Statistical analysis was performed by two-way ANOVA; *** and **** signify P < 0.001 and P < 0.0001, respectively. (G) AlphaFold3 model of the PSKH1 complex with UNC119B (charcoal). PSKH1 kinase domain is colored gray and the C-terminal flanking helices in dark gray. PAE plot is shown in SI Appendix, Fig. S4C.

Fig. 5.

Allosteric binders converge on common sites on PSKH1. (A) Orthogonal views of PSKH1 with sites bound by Calmodulin (blue), RCN3 (yellow), UNC119B (red), and all interactors (purple) highlighted. (B) Schematic overview of positive and negative allosteric binding interactions identified in this work. Extremes in Ca²⁺ flux are predicted to dictate whether PSKH1 is activated (Calmodulin; low Ca²⁺) or inhibited (RCN1 or RCN3; high Ca²⁺) by different Ca²⁺ sensor proteins. Like Ca²⁺ sensor proteins, UNC119B binds directly to the PSKH1 kinase domain, but allosterically activates kinase activity in a Ca²⁺ and lipidation-independent manner.