PAK1 is the first discovered member1
of the mammalian PAK (p21-activated kinase) family, which comprises six proteins divided in group I (PAK1–3) and group II (PAK4–6) on the basis of their structural and functional features.2-6
We will focus on the prototype PAK1 as its implication in cancer is well established and represents an ideal target for future personalized oncology treatments. Moreover its dynamics have been extensively investigated by various cell imaging approaches.
The biological functions of PAK1 are disparate and include actin dynamics, cell motility, cell cycle progression, cell division, gene transcription, cell proliferation and apoptosis.2-6
However, it is still poorly understood how at molecular level PAK1 can perform such a variety of functions. A very reasonable assumption is that the fine control of localization and activation of PAK1 is the mechanism used by the cell to activate the right PAK1-dependent pathway according to the cell cycle status or in response to extracellular stimuli.
PAK1 is a downstream effector of the Rho-family GTPases, Rac1 and Cdc42. As all PAK proteins, PAK1 consists of a highly conserved C-terminal catalytic kinase domain and an N-terminal region with a regulatory role. The PAK1 regulatory domain contains (1) a GTPase-binding domain (GBD), (2) an auto-inhibitory switch domain (IS) and (3) several Pro-rich motifs that bind to SH3 domains of Nck and Grb2 adapters or of the PIX α/β exchange factors. Inactive PAK1 has a homodimeric trans-inhibited conformation, in which the N-terminal inhibitory IS domain of one PAK1 molecule binds and inhibits the catalytic domain of the other one in the dimer.7
When GTP-loaded Rac1 or Cdc42 (the activators) bind to the N-terminal GBD domain, a series of PAK1 conformational changes is triggered, leading to disruption of dimerization, removal of the trans-inhibitory interactions and consequent acquisition of an active state for the kinase C-terminal domain.7-9
PAK1 activation by Rac1 and Cdc42 has been well established and biochemically characterized,10
but this simple Rac1/Cdc42-PAK1 pathway explains only very partially the spatiotemporal regulation and the multiple cellular roles of PAK1. At least two levels of complexity need to be taken into account. First, there are also GTPase-independent mechanisms that regulate PAK1 kinase activity, such as phosphorylation by other kinases (including Etk,11,12
), or interactions with other proteins; among the PAK1 partners, beside the already mentioned SH3-containing Nck, Grb2 and α/βPIX, it is worth mentioning the tumor suppressor Merlin16,17
and the integrin-binding protein Nischarin18
that both inhibit the PAK1 kinase activity. Second, PAK1 has kinase-independent functions that have been ascribed to its scaffold capacity,19-21
i.e., PAK1 in some cases acts not by phosphorylating targets but by facilitating the assembly of multi-protein signaling modules.
In the past decade, the live cell imaging came in help of classical approaches, based on biochemistry techniques and immuno-fluorescence studies. Thanks to the developments of fluorescent protein fusions and of automated video-microscopes, we start to have the right tools to decipher the dynamics of PAK1 at high resolution, both temporally and spatially. Importantly, since PAK1 is normally auto-inhibited, it is essential to know not only where/when PAK1 localizes, but also where/when PAK1 is activated.