Abstract
The continuous assembly and disassembly of focal adhesions is required for efficient cell spreading and migration. The G-protein-coupled receptor kinase-interacting protein 1 (GIT1) is a multidomain protein whose dynamic localization to sites of cytoskeletal remodeling is critically involved in the regulation of these processes. Here we provide evidence that the subcellular localization of GIT1 is regulated by protein kinase D3 (PKD3) through direct phosphorylation on serine 46. GIT1 phosphorylation on serine 46 was abrograted by PKD3 depletion, thereby identifying GIT1 as the first specific substrate for this kinase. A GIT1 S46D phosphomimetic mutant localized to motile, paxillin-positive cytoplasmic complexes, whereas the phosphorylation-deficient GIT1 S46A was enriched in focal adhesions. We propose that phosphorylation of GIT1 on serine 46 by PKD3 represents a molecular switch by which GIT1 localization, paxillin trafficking, and cellular protrusive activity are regulated.
Highlights
The multidomain protein G-protein-coupled receptor kinase-interacting protein 1 (GIT1) is involved in cytoskeletal rearrangements and cell motility
A GIT1 S46D phosphomimetic mutant localized to motile, paxillin-positive cytoplasmic complexes, whereas the phosphorylation-deficient GIT1 S46A was enriched in focal adhesions
In this study we identify and characterize the function of a novel phosphorylation site within GIT1 on serine 46
Summary
The multidomain protein GIT1 is involved in cytoskeletal rearrangements and cell motility. GIT1 was found to cycle between the leading edge and FAs in motile punctate cytoplasmic structures [10] These structures were described as supramolecular protein complexes containing paxillin, PIX, and PAK that were not associated with any membrane markers [10]. At the Golgi complex, PKD regulates vesicular traffic to the plasma membrane, whereas at the plasma membrane PKD is involved in the regulation of cell shape, movement, and invasion In line with such functions, substrates associated with actin cytoskeletal remodeling including cortactin, DLC1, RIN1, SSH1L, and Pak were recently identified to be phosphorylated by PKD [21,22,23,24,25,26]. We provide evidence that the cellular activity of GIT1 via serine 46 phosphorylation is exclusively regulated by PKD3, identifying GIT1 as the first specific substrate of this particular PKD isoform
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