Control of atypical PKCι membrane dissociation by tyrosine phosphorylation within a PB1-C1 interdomain interface

Abstract

Atypical PKCs are cell polarity kinases that operate at the plasma membrane where they function within multiple molecular complexes to contribute to the establishment and maintenance of polarity. In contrast to the classical and novel PKCs, atypical PKCs do not respond to diacylglycerol cues to bind the membrane compartment. Until recently, it was not clear how aPKCs are recruited; whether aPKCs can directly interact with membranes or whether they are dependent on other protein interactors to do so. Two recent studies identified the pseudosubstrate region and the C1 domain as direct membrane interaction modules; however, their relative importance and coupling are unknown. We combined molecular modeling and functional assays to show that the regulatory module of aPKCι, comprising the PB1 pseudosubstrate and C1 domains, forms a cooperative and spatially continuous invariant membrane interaction platform. Furthermore, we show the coordinated orientation of membrane-binding elements within the regulatory module requires a key PB1-C1 interfacial β-strand (beta-strand linker). We show this element contains a highly conserved Tyr residue that can be phosphorylated and that negatively regulates the integrity of the regulatory module, leading to membrane release. We thus expose a hitherto unknown regulatory mechanism of aPKCι membrane binding and release during cell polarization.

Keywords: atypical protein kinase C; cell polarity; cell signaling; membrane recruitment; tyrosine phosphorylation.

Figure 1

The aPKCι regulatory module contains a continuous membrane-binding surface and is localized to mitotic cell membranes.A, representation of the aPKCι regulatory module as predicted by Alphafold Colab (best scoring model). The individual domains and regions are indicated and colored; from N-C PB1 domain (cyan), pseudosubstrate (PS) region (dark purple), beta-strand linker (BSL) (light purple), C1 domain (blue). B, per residue prediction score of the MODA prediction with above-threshold scoring regions indicated. C, cartoon diagram (omitting secondary structure features) of predicted membrane interacting regions from the MODA prediction displayed in the AF Colab model. D, mesh representation of panel C showing a continuous sidechain surface. E, localization of FLAG-tagged aPKCι regulatory module (RM) in HEK293T cells; red asterisks indicate mitotic cells. F, localization of FLAG-tagged aPKCι RM in nocodazole-treated (500 nM, 16 h) HEK293T cells. MODA, Membrane Optimal Docking Algorithm.

Figure 2

Probing mutations within the aPKCι^RM on membrane binding in mitotic cells reveal a role for both the PS and C1 domain.A, localization of WT FLAG-tagged regulatory module (RM) in nocodazole-treated (500 nM, 16 h) HEK293T cells. B–E, expression of the indicated mutant FLAG-tagged regulatory modules in nocodazole-treated (500 nM, 16 h) HEK293T cells. Mutated residues are depicted on the protein model in red. F, quantification of membrane/cytoplasmic pixel intensities measured in >45 cells imaged via confocal microscopy. Representative experiment of three biological replicates. Differences analyzed via one-way ANOVA (∗∗∗∗p ≤ 0.0001).

Figure 3

Tyrosine-136 phosphorylation within the BSL affects aPKCι stability with a minor impact on catalytic activity.A, identification of main phosphorylation sites on aPKCι identified in the Phosphosite database. B, visualization of Tyr-136 central to the BSL region of the regulatory module, color coded as in Fig. 1A. C, peptide-pulldown of Myc-tagged aPKCι kinase domain with extended pseudosubstrate peptides (Biotin-Ahx-SIYRRGARRWRKLYCANGHT-CONH2) containing the indicated modifications at Y¹⁴, i.e., pY, Y/E and Y/F. D, co-expression of FLAG-tagged regulatory module and Myc-tagged kinase domain of aPKC in HEK293T cells. Equal amounts of DNA were transfected and stabilization of Myc-tagged kinase domain by different mutants of the regulatory module was followed. Quantification of Myc-kinase domain levels relative to WT aPKCι^RM co-expression shown in the right panel (n = 4 biological replicates, average ± SD, analyzed via one-sample t test (v.a.v. a value of 1): ns p > 0.05, ∗p ≤ 0.05, ∗∗∗p ≤ 0.001). E, DSF assay measuring thermal stability of WT and mutant fl-aPKCι in absence or presence of ATP.Mg²⁺. Plotted is the T_m derived from the inflection point of the melting curve (using the derivative maximum) (average ± SD, analyzed via one-way ANOVA: ns p > 0.05, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001). F, in vitro kinase assay with immunoprecipitated WT or mutant kinases on MBP. Incorporation of new phosphates into MBP was followed with ATP-γS. Quantification of MBP thiophosphate levels shown on the right (n = 4 biological replicates, average ± SD, analyzed via unpaired t test: ns p > 0.05). MBP, myelin basic protein; PKC, protein kinase C.

Figure 4

Tyrosine-136 phosphorylation within the BSL correlates with cytoplasmic aPKCι^RM.A–E, expression of the indicated mutant FLAG-tagged regulatory modules (RM) in nocodazole-treated (500 nM, 16 h) HEK293T cells. F, quantification of membrane/cytoplasmic pixel intensities measured in >45 cells imaged via confocal microscopy. Representative experiment of three biological replicates. Differences analyzed via one-way ANOVA (ns p > 0.05, ∗∗∗∗p ≤ 0.0001). G, Western blot analysis of phospho-Tyr-136 levels in the in untreated (mixed pop.) cells or cells treated with 500 nM nocodazole for 16 h (Noc.). H, representation of the observed localization of regulatory module species and possible mapping of its phospho-form. I, fractionation of cells by ultracentrifugation and probing for Tyr-136 phosphorylation of the regulatory module (representative experiment of n = 2 biological replicates).

Figure 5

Src phosphorylates Tyr-136 and influences aPKCι^RM localization.A, effect of Src inhibition on Tyr-136 phosphorylation. HEK293T cells expressing FLAG-tagged regulatory module were treated with nocodazole (500 nM, 16 h) or left untreated and incubated with the indicated inhibitors (PP2 10 μM, dasatinib 1 μM or KX2-931 1 μM) for 1 h before lysis and probed for Tyr-136 phosphorylation. Quantification of pY136 levels shown in the right panel (n = 3, average ± SD) analyzed via one-sample t test (v.a.v. a value of 1; ∗p ≤ 0.05, ∗∗p ≤ 0.01); B, in vitro phosphorylation of the aPKCι^RM with Src. WT or Y136 F aPKCι^RM was immunoprecipitated from HEK293T cells and incubated with Src (20 ng) and ATP.Mg²⁺ for 20 min after which the reactions were detected for Tyr-136 phosphorylation. C, levels of Tyr-136 phosphorylation upon co-expression of aPKCι^RM with Src-HA in HEK293T cells treated with nocodazole or left untreated. D, localization of aPKCι^RM upon co-expression with Src-HA in nocodazole (500 nM, 16 h) treated HEK293T cells. Mitotic cells with high levels of Src-HA display increased cytoplasmic staining for aPKCι^RM compared to cells that only express aPKCι^RM. (The red asterisk indicates an interphase cell where aPKCι^RM is nuclear). E, quantification of membrane/cytoplasmic FLAG pixel intensities plotted against the HA pixel intensities in 46 cells imaged via confocal microscopy. PKC, protein kinase C; RM, regulatory modules.

Figure 6

Lipid-binding preferences of aPKCι mutants and model for the effects of Tyr-136 phosphorylation compared to PS disengagement.A, silver-stained SDS-PAGE gel of the Myc-aPKC-2xStrep-Par6 binary complexes purified through 2xStrep-Par6. B, lipid overlay assay using the protein complexes defined in (A). Levels of aPKCι bound to the PIP-strip membrane were quantified using anti-Myc tag antibody. C, schematic representation of the impact of mutants and phospho-states of aPKCι. The left panel displays the anticipated consequence of Tyr-136 phosphorylation in the BSL as mimicked by a Y136E substitution, disrupting the continuous membrane binding platform constituted by the PS and C1 domain. The right panel displays the impact of a pseudo-substrate disengaged conformation, mimicked by an A129E substitution which mainly results in relieved auto-inhibition and increased catalysis. BSL, beta-strand linker; PS, pseudosubstrate; PKC, protein kinase C.