P21-activated kinases (Paks), a family of serine/threonine kinases, are effectors of the Rho GTPases Cdc42 and Rac1. Mammalian Pak1 and Pak homologs in simple eukaryotes are implicated in controlling G2/M transition and/or mitosis. Another serine/threonine kinase, polo-like kinase 1 (Plk1), is an important regulator of mitotic events, such as centrosome maturation, mitotic entry, spindle formation, sister chromatid cohesion and cytokinesis. Plk1 phosphorylation is thought to be one of the critical regulatory events leading to these Plk1-mediated functions. We show here that Pak1 is required for cell proliferation, mitotic progression and Plk1 activity in HeLa cells. Gain or loss of Pak function directly impacted phosphorylation and activity of Plk1. Phosphorylation of Plk1 on Ser 49 is important for metaphase-associated events. Inhibition of Pak activity leads to delay in G2/M progression and abnormal spindle formation, mirroring some attributes of Plk1 deregulation. Our results reveal a role for Pak in regulating Plk1 activity and mitotic progression, and connect Pak to the complex protein interaction network enabling cell division.

Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3–Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth.