Abstract
BackgroundProtein kinase D (PKD) enzymes regulate cofilin-driven actin reorganization and directed cell migration through both p21-activated kinase 4 (PAK4) and the phosphatase slingshot 1L (SSH1L). The relative contributions of different endogenous PKD isoforms to both signaling pathways have not been elucidated, sufficiently.Methodology/Principal FindingsWe here analyzed two cell lines (HeLa and MDA-MB-468) that express the subtypes protein kinase D2 (PKD2) and protein kinase D3 (PKD3). We show that under normal growth conditions both isoforms can form a complex, in which PKD3 is basally-active and PKD2 is inactive. Basal activity of PKD3 mediates PAK4 activity and downstream signaling, but does not significantly inhibit SSH1L. This signaling constellation was required for facilitating directed cell migration. Activation of PKD2 and further increase of PKD3 activity leads to additional phosphorylation and inhibition of endogenous SSH1L. Net effect is a dramatic increase in phospho-cofilin and a decrease in cell migration, since now both PAK4 and SSH1L are regulated by the active PKD2/PKD3 complex.Conclusions/SignificanceOur data suggest that PKD complexes provide an interface for both cofilin regulatory pathways. Dependent on the activity of involved PKD enzymes signaling can be balanced to guarantee a functional cofilin activity cycle and increase cell migration, or imbalanced to decrease cell migration. Our data also provide an explanation of how PKD isoforms mediate different effects on directed cell migration.
Highlights
In order to migrate towards a chemotactic stimulus cells activate cofilin at the leading edge [1,2,3]
Results obtained with RT-PCR were confirmed by immunoprecipitations of endogenous PKD1, protein kinase D2 (PKD2) or protein kinase D3 (PKD3) using subtype-specific antibodies (Fig. 1B, bottom panels)
To test if such complexes can be formed in cells by endogenous proteins we immunoprecipitated endogenous PKD2 or PKD3 using subtype specific antibodies, and probed for the other isoform, respectively
Summary
In order to migrate towards a chemotactic stimulus cells activate cofilin at the leading edge [1,2,3]. Phosphorylation of S3 leads to cofilin inactivation and is mediated by LIM domain kinase (LIMK), whereas the phosphatase slingshot 1L (SSH1L) dephosphorylates this site [3]. Both regulatory enzymes, LIMK and SSH1L guarantee a functional cofilin activity cycle (cyclic activation and reactivation of cofilin to facilitate Factin reorganization processes) at the leading edge of cells [5]. Protein kinase D (PKD) enzymes regulate cofilin-driven actin reorganization and directed cell migration through both p21-activated kinase 4 (PAK4) and the phosphatase slingshot 1L (SSH1L). The relative contributions of different endogenous PKD isoforms to both signaling pathways have not been elucidated, sufficiently
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