Aim. This study aimed to evaluate how S6K1 isoform expression affects the activities of Akt1, AMPK, PKC, and PKA, as well as their related kinase signaling pathways. Methods. MCF-7 cells and a subline with disrupted p70 and p85 S6K1 isoform expression (MCF7/p60+/p70–/p85–) were cultured under different growth conditions. Western blot analysis was performed on cell lysates using antibodies specific to phosphorylated substrates of Akt1, AMPK, PKC, PKA, and GSK-3β/pSer9. Results. The phosphorylation pattern of kinase substrates showed that changes in S6K1 isoform expression influenced either the activity of Akt1, AMPK, PKC, PKA, or their substrate specificity. Unexpectedly, in the MCF-7 subline exhibiting EMT features, GSK-3β kinase – an Akt1 substrate and EMT inhibitor – was not downregulated by phosphorylation of Ser9. Conclusions. S6K1, a ribosomal protein kinase involved in EMT regulation, can modulate the activity of Akt1, AMPK, PKC, PKA, and GSK-3β, as well as their substrate specificity; therefore, all these kinases may be involved in EMT regulation. GSK-3β could also have a bifunctional role in EMT progression initiated by changes in S6K1 isoform expression that modulate its activity.