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Paradoxical non-catalytic kinase functions are driven by inhibitor-induced displacement of autoinhibitory domains

ABSTRACT: Protein kinases are crucial regulators of cellular processes, and their dysregulation is frequently associated with human disorders. ATP-competitive type I inhibitors are widely used to block aberrant kinase activity. However, their effect on non-catalytic kinase functions such as conformation and protein-protein interactions (PPIs) remain largely uncharacterized. Here we present a multi-proteomics strategy to systematically measure these changes. First, we developed affinity purification (AP) and limited proteolysis coupled to mass spectrometry (AP-LiP-MS) to measure structural changes at high sequence coverage. We benchmarked AP-LiP-MS using DCLK1 before analyzing structural changes upon probe binding to CAMKK2, CHEK1, and PRKCA. All kinases underwent structural changes consistent with dissociation of domain-domain interactions (DDIs) between the kinase domain (KD) and the autoinhibitory domain (AID). Next, we applied AP and in vivo proximity labeling to analyze changes in kinase PPI networks. This revealed extensive and characteristic rewiring of functionally important PPIs, independent of catalytic activity. For instance, SCG-CAMKK2-1 binding to CAMKK2 sequestered PRKAA1, inhibiting its regulation by other kinases, while rabusertib caused CLPB-CHEK1 dissociation and mitochondrial fragmentation, and Gö 6983 resulted in PRKCA recruitment to cellular junctions. We propose that these unexpected phenotypes originate from on-target, off-mechanism effects, driven by structural changes and PPI rewiring. We advocate for the use of our workflow to systematically characterize overlooked consequences of small-molecule binding during drug development.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Early Embryonic Cell

SUBMITTER: Viviane Reber

LAB HEAD: Matthias Gstaiger

PROVIDER: PXD069830 | Pride | 2026-06-17

REPOSITORIES: Pride

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			Action	DRS
	200320_human_BirA_iRT_GFP_streptavidin.fasta	Fasta
	200320_human_BirA_iRT_GFP_streptavidin_no_CAMKK2.fasta	Fasta
	200320_human_BirA_iRT_GFP_streptavidin_no_PRKCA.fasta	Fasta
	200320_human_PK_iRT_tag_GFP_streptavidin.fasta	Fasta
	200320_human_PK_iRT_tag_GFP_streptavidin_no_CAMKK2.fasta	Fasta

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Project description:Background: Negative-pressure wound therapy has become widely available in modern chronic wound cares. Accelerated keratinocyte (HaCaT cell) movements and decreased E-cadherin expressions induced by the applied negative pressure gradient of 125 mmHg (NP) have been reported in previous studies. However, the molecular mechanism for E-cadherin regulations under NP remains unexplored. We highlighted the signal transduction involved in NP-induced E-cadherin regulation for keratinocytes in the study. Results: Microarray results showed that catenin 1 (CTNND1) gene, the gene encoding the p120-catenin (p120ctn) in cell-cell junctions, was significantly (p = 0.0005) upregulated in HaCaT cells under NP for 12 hours in comparison with those at ambient pressure (AP). Cell fractionations and immunoblotting data showed that NP increased p120ctn phosphorylation, and resulted in p120ctn translocation from the plasma membrane to the cytoplasm. Fluorescent stains suggested that NP diminished the co-localization of p120ctn and E-cadherin on the plasma membrane. Similar phenomenon was also observed in the cells with overexpressed p120ctn at NP. Interestingly, overexpression of dN- p120ctn, a deletion mutant of p120ctn without the N-terminal phosphorylation sites, restored the adherens junctions (AJ) at NP. Knockdown of p120ctn with lentiviral small hairpin RNA not only diminished E-cadherin expressions but also accelerated cell movement at AP. Conclusions: Phosphorylation of p120ctn is endowed to respond rapidly to NP and contributes to the AJ disassembly. This NP-induced E-cadherin downregulation can possibly accelerate wound-healing process. Conclusions: Phosphorylation of p120ctn is endowed to respond rapidly to NP and contributes to the AJ disassembly. This NP-induced E-cadherin downregulation can possibly accelerate wound-healing process. HaCaT cells under negative pressure (NP) gradient of 125 mmHg for 12 hours in comparison with those at ambient pressure (AP) were tested for RNA extraction and hybridization on Affymetrix microarrays. The experiments have been biological replicated three times. The differential expression gene between NP and AP were selected and validated with qPCR method.

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Paradoxical non-catalytic kinase functions are driven by inhibitor-induced displacement of autoinhibitory domains

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