The essential role of Akt in cancer cell growth and survival has made it an attractive target for the development of anti-cancer therapeutics (Hennessy et al., 2005
). However recent studies that have revealed opposing functions of Akt1 and Akt2 in the regulation of carcinoma migration leading to metastatic dissemination have necessitated a reevaluation of inhibition of Akt in cancer therapy (Chin and Toker, 2009
; Sawyers, 2006
). Thus far, Akt isoform-specific substrates and molecular mechanisms that are responsible for this distinction have remained elusive. Indeed, despite over 150 Akt substrates that have been characterized to date, only a few have been evaluated for isoform specificity. These include the cell cycle regulators p21 CIP1 (Heron-Milhavet et al., 2006
) and SKP2 (Gao et al., 2009
) that are Akt1-specific targets, whereas MDM2 (Brognard et al., 2007
) and AS160 (Bouzakri et al., 2006
; Gonzalez and McGraw, 2009
) are specifically phosphorylated by Akt2. However, none of these account for the differential effects of Akt isoforms on invasive migration.
In the present study we have identified palladin, an actin-associated protein, as an Akt1 substrate. Phosphorylation of palladin by PI 3-K and Akt1 signaling by physiological stimuli such as IGF-1 as well as by genetic mutations in the pathway, such as PTEN loss and oncogenic PIK3CA supports a role for this Akt1 target in both physiological and pathophysiological signaling. The results presented here also provide evidence for a functional role for palladin in invasive migration of breast cancer cells, and highlight the critical role of palladin phosphorylation in regulating migration and actin bundling.
Despite the high sequence and structural homology among Akt isoforms, Akt1 but not Akt2 interacts with and phosphorylates palladin. Substrate selectivity of isoforms can be achieved by several potential mechanisms, including subcellular compartmentalization, binding to distinct scaffolding molecules and differential activation or regulation by extracellular stimuli. Indeed, it has recently been shown that in insulin-stimulated adipocytes Akt2 preferentially accumulates at the plasma membrane and specifically phosphorylates the substrate AS160 to regulate GLUT4 trafficking (Gonzalez and McGraw, 2009
). However, differential compartmentalization of Akt isoforms is unlikely to account for the exclusivity of palladin phosphorylation by Akt1, since purified Akt1 but not Akt2 phosphorylates palladin in a cell-free system. It is more likely that an intrinsic molecular determinant or ‘docking motif’ on Akt1 promotes its association with palladin. The data from the Akt chimeras studies indicate that the linker region of Akt1 plays an important role in determining the isoform-specificity of palladin phosphorylation. Interestingly, Field and colleagues have identified the same region as an important determinant in the ability of Akt1 and Akt2 to form dorsal ruffles during fibroblast migration (Zhou et al., 2006
). The precise residues within the linker region that are responsible for this isoform-specificity are yet to be determined. Regardless, these data point to an important function of the linker region in modulating palladin phosphorylation by the Akt1 pathway.
Our findings that palladin has an anti-migratory role in breast cancer cells differ from a recent study in which palladin knockdown by siRNA inhibited invasive migration in Transwell assays (Goicoechea et al., 2009
). The distinction between the two studies could be explained by the different migration conditions or chemoattractants used. In the present study results obtained with Transwell assays were corroborated using time-lapse video microscopy as well as 3-D cultures. In all cases, cells expressing palladin shRNA exhibited highly migratory and invasive phenotypes in four distinct breast cancer cell lines. Most importantly, rescue experiments revealed that overexpression of palladin is able to reverse the migratory effect induced by palladin shRNA, further demonstrating the specificity of the approach and confirming the inhibitory function of palladin phosphorylation on invasive migration of breast cancer cells.
Our studies also point to a new mechanism by which palladin phosphorylation modulates its actin regulatory activity. We show that phosphorylation of palladin promotes actin bundling, consistent with the observation that the Akt motif at Ser507 resides in a domain that critical for cross-linking actin (Dixon et al., 2008
). We propose that by promoting the formation of actin bundles and re-organizing the actin cytoskeleton, palladin phosphorylation inhibits cell migration. It is also worth noting that a link between actin filaments and integrin receptors plays a crucial role in dictating the organization and stability of adhesions during cell migration (Vicente-Manzanares et al., 2009
). Polymerization of actin fibers regulates clustering of activated β1 integrins at the leading edge of migrating fibroblasts (Galbraith et al., 2007
). Interestingly, palladin can stabilize β1 integrins in fibroblasts (Liu et al., 2007
). Since palladin phosphorylation modulates the organization of actin cytoskeleton, it is possible that Ser507 phosphorylation by Akt1 could regulate β1 integrin signaling to the migration phenotype.
Coordinated up-regulation of both the stimulatory and inhibitory branch of actin motility machinery has been observed in invasive carcinoma cells (Wang et al., 2004
). Accordingly, expression of palladin is increased in human breast tumor tissues (Goicoechea et al., 2009
) and invasive mammary tumors in rats (Wang et al., 2004
). Since our data show that palladin phosphorylation functions to inhibit invasive migration, it will be interesting to determine the relative phosphorylation levels of palladin in breast tumor tissues. This will only be possible with the use of specific phospho Ser507 palladin antibodies.
In summary, the present study defines a mechanism that links palladin to Akt1-mediated inhibition of breast cancer cell migration. We show that Akt1, but not Akt2, phosphorylates palladin at Ser507. In turn this induces reorganization of the actin cytoskeleton through actin bundling activity and ultimately leads to inhibition of migration of breast cancer cells. To our knowledge, this is the first specific substrate that accounts for the distinct roles of Akt isoforms in cell migration. Undoubtedly, more isoform-specific substrates that confer functional selectivity remain to be identified. Given the fact that Akt isoforms are frequently hyperactivated in breast cancer and that considerable effort is underway to develop pharmacological Akt inhibitors as therapeutic agents, these findings underscore the importance of dissecting the precise mechanisms by which the PI 3-K and Akt pathway regulates breast cancer invasive migration leading to metastatic dissemination.