Kinase play-off on lipid turf hints at a molecular basis for new-found tumor-suppressor activity of PKCs

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Migration of cells mediates key life processes starting from the beginning of life, through development and adulthood (1). This essential process is a teleological culmination of a complex molecular circuitry that generates signals that propagate like waves to orchestrate cytoskeletal rearrangements necessary for cell motion. To set the stage for discussing the role of kinases, which are proteins that transfer phosphate moieties to bio-molecular substrates, and are putative “star” players in cell migration, let us attempt to create a simplistic mental sketch of this intricate molecular interplay. The major groups of molecular players based on the current consensus model of cell motion are (1) external cues, which are molecular gradients that determine the overall direction of the movement; (2) signal-receiving interface at the cell boundary, composed of lipids and lipid-interacting proteins, especially kinases; (3) proteins, messenger molecules, and ions that transmit the signal to induce requisite cytoskeletal rearrangements at all steps of migration. The lipid-protein interface receives the external signal and polarizes the cell. This establishes a cellular front-rear gradient of molecular factors that regulate the migration cycle, which involves the formation of protrusions followed by adhesion to the extracellular matrix and subsequent detachment (1).

A fine balance between the activities of molecular factors regulating cell migration is necessary to maintain homeostasis. Conversely, disruptions in molecular regulation of cell migration are linked to severe pathophysiological consequences, including inflammatory diseases, vascular disorders, and cancer (1). Hence, there has been significant interest in manipulating the activity of key molecular players of cell migration to develop therapeutic interventions for cancer (2). While the end goal has been clear for the past four decades, achieving a successful therapeutic intervention for cancer by developing substrates that can alter the activity of molecular regulators of cell migration remains elusive. This is because, despite tremendous progress in understanding fundamental molecular mechanisms underlying cell migration, many questions remain unanswered. As an example, kinases, which are major cancer therapeutic targets, have not led to successful cancer treatments when either inhibited or activated (2). Why is that? How do we identify the role of a kinase in cell migration to select the correct therapeutic intervention? Relevant to our discussion on kinases is the case of protein kinase C (PKC) proteins. Based on a discovery in 1982 that PKCs can be activated upon binding to tumor-promoting molecules, it was assumed that inhibition of PKCs could lead to an effective cancer treatment strategy (3). The association of PKCs with cancer was hence a topic of intense research in both academia and pharma (4). However, a series of recent studies provided opposing evidence indicating that there was in fact a loss of function of PKCs in several cancer-associated PKC mutations (5,6). These studies hinted at a role reversal of PKCs as tumor suppressors instead of promoters. Most importantly, the PKC story showed that the roles of kinases in cell migration might be context dependent. What clearly emerged from this dichotomy was the urgent need for detailed molecular-level investigations on the interplay of kinases with other regulators of cell migration and also with other kinases.

In this issue of Biophysical Journal, Gordon, Falke, and colleagues applied single-molecule studies to investigate the interactions of three key kinases involved in cell migration and proliferation in the backdrop of model supported lipid bilayers (SLBs) (7). Specifically, they evaluated the interactions of a PKC isozyme (PKCα), 3-phosphoinositide-dependent protein kinase (PDK1), and RAC-alpha serine/threonine protein kinase (AKT1).

PDK1/AKT1 interaction is a critical signaling step that drives downstream pathways necessary for cell migration and proliferation (8). At the molecular level, the interaction between PDK1 and AKT1 involves the phosphorylation of AKT1 by PDK1. This requires the recruitment of PDK1 molecules close to AKT1 molecules. This is achieved by the lipid “turf” which brings these key migration players in proximity. The lipid phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), which is a signaling lipid that promotes cell growth and migration, is transiently present on cell membranes with resting concentrations as low as 50 nM. Upon stimulation by migratory and proliferation factors, its levels can increase ∼50 times (9). Both PDK1 and AKT1 have binding sites for PIP3. Hence, when PIP3 levels increase in the cell membrane, both kinases are recruited to the membrane, which increases their local concentration and allows the phosphorylation of AKT1 by PDK1 activating downstream signaling necessary for cell migration. An earlier study by the Falke laboratory had provided evidence for a molecular pathway in which the recruitment of PKCα to the cell membrane would lead to an increase in the levels of the growth mediator PIP3, which would in turn lead to the initiation of the PDK1/AKT1 signaling pathway, thus justifying a cell migration-promoting role for PKCα (8).

In their current article, the authors recorded the diffusion of the three kinase molecules on SLBs (7). When a fluorescently tagged kinase binds to its lipid-binding partner on an SLB, its diffusivity decreases, allowing visualization by single-molecule total internal reflection microscopy (smTIRFM). The diffusivity is expected to lower further when proximal kinases bind to form heterodimers on the lipid bilayer. Based on this premise, the results obtained by the authors showed that both PDK1 and AKT1 were recruited to SLBs containing 2 mol % PIP3. PKCα, on the other hand, was recruited to membranes containing another anionic phospholipid, phosphatidylserine (PS). The diffusion constants of PDK1 in the presence of AKT1 and PKCα were first measured separately. By plotting the diffusion constants of PDK1 with increasing concentrations of either AKT1 or PKCα, the authors determined that the affinity of a PDK1:PKCα heterodimer was eight times higher than the affinity of a PDK1:AKT1 heterodimer. A key experiment in the article was a single-molecule competition experiment between the three kinases. When a saturating concentration of PKCα was added to fluorescent PDK1/AKT1 heterodimers formed on an SLB, the diffusion constant value of the fluorescent molecules on the SLB changed to match the diffusion constant of the PDK1/PKCα heterodimer. This result indicated that PKCα could displace AKT1 from the PDK1/AKT1 heterodimer (Fig. 1). Therefore, increased levels of PKCα might be able to block the activation of AKT1 by PDK1, thereby inhibiting downstream pathways that activate cell proliferation and migration (Fig. 1). Consequently, this elegant single-molecule experiment provided a plausible molecular interaction pathway to explain the role reversal of PKCα from a tumor promoter to a suppressor. Importantly, the experiment was performed at physiologically relevant kinase concentrations, making the results pertinent in the context of cell migration.

Figure 1.

Cartoon representation of the seminal smTIRFM experiment set up by Gordon et al. to decipher interactions between three key kinases involved in regulation of cell migration and proliferation. The displacement of AKT1 from PDK1/AKT1 heterodimer by PKCα might explain the tumor-suppressor role of PKCα. Lipid bilayer image credit Crevis/Shutterstock.com.

The conclusions drawn from the article form bold implications to the mechanistic understanding of the role of kinases in cell migration. While careful setups of single-molecule studies in artificial systems are invaluable in teasing apart specific interactions between the molecular players of biologically intricate processes, it is important to note that results from these studies ultimately lead to mechanistic hypotheses. Models proposed on the basis of single-molecule studies in controlled SLB systems need validation in multiple levels with increased biological complexity, including studies in cellular systems. Possible direct regulatory roles of the lipid turf that forms the backdrop of single-molecule studies on kinases in this article (7) are also becoming increasingly evident. Hence, new tools (10) and methods to simultaneously track the activities of lipid (11) and protein players in the living milieu is the next challenging, albeit exciting, endeavor toward creating a molecular picture of cell migration.

Editor: Sudipta Maiti