Roselova, P. | bioRxiv Science

bioRxiv2021

PAK1 and PAK2 in the cell metabolism regulation

P21-activated kinases (PAKs) regulate processes associated with cytoskeletal rearrangements, such as cell division, adhesion, and migration. The possible regulatory role of PAKs in cell metabolism has not been well explored, but increasing evidence suggests that a cell metabolic phenotype is related to cell interactions with the microenvironment. We analyzed the impact of PAK inhibition by small molecule inhibitors, small interfering RNA, or gene knockout on the rates of mitochondrial respiration and aerobic glycolysis. Pharmacological inhibition of PAK group I by IPA-3 induced a strong decrease in metabolic rates in human adherent cancer cell lines, leukemia/lymphoma cell lines, and primary leukemia cells. The immediate effect of FRAX597, which inhibits PAK kinase activity, was moderate, indicating that PAK nonkinase functions are essential for cell metabolism. Selective downregulation or deletion of PAK2 was associated with a shift toward oxidative phosphorylation. Alternatively, PAK1 knockout resulted in increased glycolysis. However, the overall metabolic capacity was not substantially reduced by PAK1 or PAK2 deletion, possibly due to partial redundancy in PAK1/PAK2 regulatory roles or to activation of other compensatory mechanisms.

cell biology↗

bioRxiv2020

Group I p21-activated kinases in leukemia cell adhesion to fibronectin

P21-activated kinases (PAK) regulate many processes associated with cytoskeleton dynamics, including cell adhesion, migration, and apoptosis. PAK function is frequently altered in cancer, and PAK were proposed as therapy targets both in solid tumors and in hematological malignancies. However, current knowledge about PAK function in cell adhesion is mainly based on adherent cell models. Moreover, existing functional differences among the individual PAK family members are unsufficiently characterized. We measured expression of PAK group I members in leukemia cell lines and in primary leukemia cells, both on protein and mRNA levels. In functional assays, we analyzed the effect of two PAK inhibitors with different mechanisms of action, IPA-3 and FRAX597. Changes in cell interaction with fibronectin were monitored through impedance measurement and by interference reflection microscopy. Cytotoxic effects of inhibitors were assessed by Annexin V/propidium iodide test. PAK intracellular localization was analyzed by confocal microscopy. PAK2 transcript was dominant in cell lines, whereas primary leukemia cells also expressed comparable amount of PAK1, which was detected as two transcription isoforms: PAK1-full and PAK1{Delta}15. PAK1{Delta}15 and PAK2 transcript levels correlated with surface density of integrins {beta}1 and V{beta}3. PAK1-full, but not PAK2, was present in membrane protrusions. The inhibitors had partly opposed effects: IPA-3, which prevents PAK activation, induced cell contraction in semi-adherent HEL cells only. FRAX597, which inhibits PAK kinase activity, increased cell-surface contact area in all leukemia cells. Both inhibitors reduced the stability of cell attachment and induced cell death. Although many cells accumulated high FRAX597 amounts, low doses were sufficient to kill sensitive cells. FRAX597-induced cell death was fast in the MV4-11 cell line and in primary AML cells. Although PAK group I seem to be essential for leukemia cell adhesion and survival, and might thus serve as therapy targets, many PAK functions still remain to be attributed to individual isoforms and to their functional domains.

cell biology↗