Panoply™ Human PIM3 Over-expressing Stable Cell Line

The proto-oncogene serine/threonine protein kinase PIM3 plays a crucial role in cancer development and progression and has been widely used as a drug target. Here, researchers investigated the quantitative changes in the cellular proteome and phosphoproteome of liver cancer cells overexpressing PIM3 to better understand its regulatory functions and potential molecular mechanisms. Signal network analysis indicated that PIM3 may coordinate multiple cellular processes, such as signal transduction, cell cycle, and apoptosis. Notably, quantitative phosphoproteomics revealed that PIM3 overexpression increased the phosphorylation levels of several Rho GTPase regulatory factors targeting RhoA, a key regulator of cell motility. Further studies confirmed that PIM3 activates RhoA, subsequently regulating cytoskeletal rearrangement and cell migration. In summary, these studies comprehensively mapped the proteome and phosphoproteome regulated by PIM3 and revealed that PIM3 promotes liver cancer cell migration and invasion by regulating the Rho GTPase signaling pathway.

Here, researchers observed increased phosphorylation levels of several GEF and GAP proteins in PIM3-overexpressing cells, such as ARHGEF5 (S57), ARHGAP1 (S27), and ARHGAP29 (S176 and S179) (Figure 1A). ARHGEF12 was the only protein with decreased phosphorylation at the S341 site (Figure 1A). Furthermore, the phosphorylation levels of several Rho GTPases identified in the phosphoproteomic analysis were further validated by immunoprecipitation (IP) and Western blotting. The Ser112 site on the Bad protein is a known phosphorylation site of PIM3, making Bad an ideal positive control. Immunoprecipitation using an anti-pSer antibody showed that the amount of Bad protein precipitated from PIM3-overexpressing cells was higher than that from the corresponding control cells. Similarly, the levels of SRGAP1, MYO9B, TIAM1, and AKAP13 proteins isolated from PIM3-overexpressing cells using an anti-pSer antibody were higher compared to the corresponding control cells, indicating that PIM3 overexpression induced increased serine phosphorylation levels of these Rho GTPase regulators (Figure 1B-E).

Figure 1. PIM3 regulates the phosphorylation of Rho GTPase modulators. (Dang Y, et al., 2020)