Fig 1: PDHA1 S152 phosphorylation by Pak1 alters the glycolytic flux in MDA-Panc 48 cells.A, Western blot to confirm the transfection of WT and S152 PDHA1 plasmids in Pak1 WT and KO MDA-Panc 48 cells. Assessment of the glycolytic flux of transfected cells using Seahorse extracellular flux (XF96) analyzer. B, graphical representation of changes in the extracellular acidification rate (ECAR, mpH/min) upon sequential addition of glucose, oligomycin, and 2-deoxy glucose (2-DG) in MDA-Panc 48 cells; followed by the ECAR values of glycolytic parameters (C) basal glycolysis, (D) glycolytic capacity, and (E) glycolytic reserve % were calculated. Each value represented as mean ± SD (n = 5); (∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < 0.0001). Pak1, p21-activated kinase; PDHA1, pyruvate dehydrogenase E1α subunit.
Fig 2: Delineating the regulatory relationship of Pak1 on PDHA1.A, coimmunoprecipitation experiment showed PDHA1 interacts with Pak1 in MDA-Panc 48 cells under physiological conditions (MDA-Panc 48 Pak1 KO cell lysate was used as the control to detect nonspecific binding). B, in vitro kinase assay performed using γ32-ATP: (B, left panel) assay performed with PDHA1 (target protein), MBP (positive control), and esterase (negative control); (B, right panel) assay performed with and without Pak1; C, phosphorylation of PDHA1 in the presence of Pak1 observed in Phos-tag gel. D, change in the PDHA1 phosphorylated protein levels upon treating the MDA-Panc 48 cells with IPA-3 inhibitor at 2 and 4 μM. E, change in the pPDHA1 levels upon restoration of constitutively active Pak1 expression transiently. MBP, myelin basic protein; Pak1, p21-activated kinase; PDHA1, pyruvate dehydrogenase E1α subunit.
Fig 3: Pak1 phosphorylates PDHA1 at Ser152 residue.A, graphical representation of sequence coverage in PDHA1 showing phosphorylation at S152. B, diagnostic ion table with the theoretical m/z values. Modifications are seen as a neutral loss on higher energy collisional dissociation fragmentation. C, mass spectra of the peptide GGCAKGKGGsMHMYAK. Inset shows the b and y ions identified from the peptide fragmentation and PAK1 consensus sequence (bold). D, ClustalW Multiple sequence alignment of PDHA1 from different species—human, rat, dove, snake, and zebrafish. The S152 (green) is conserved across species. E, Phos-tag immunoblot of PDHA1-WT and PDHA1-S152A cotransfected with PAK1 T423E. PAK1 overexpression and loading were confirmed on a parallel blot. Pak1, p21-activated kinase; PDHA1, pyruvate dehydrogenase E1α subunit.
Fig 4: Pak1 phosphorylating PDHA1 inhibits PDHA1 activity.A, MDA-Panc 48 cells treated with Pak1 inhibitor, IPA-3 (4 μM). B, transient expression of constitutively active Pak1 in MDA-Panc 48 cells Pak1 KO cells. C, PDHA1 activity in stable Pak1 clones: Pak1 KO, stable rescue, and Pak1 overexpression (Pak1 OE) cells. D, transient expression of FLAG-PDHA1 WT or phospho-null mutant (S152A) plasmid in HEK293T cells. E, transient expression of FLAG-PDHA1 WT or phosphor-mimic (S152E) plasmid in MDA-Panc 48 Pak1 KO cells. F, LDHA activity assay measured in stable Pak1 clones: Pak1 KO, Pak1 KI, and Pak1 OE cells. Data represented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, and ∗∗∗∗p < 0.0001 compared to the WT. Pak1, p21-activated kinase; PDHA1, pyruvate dehydrogenase E1α subunit.
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