Abstract
Partitioning-defective 1b (PAR1b), also known as microtubule affinity-regulating kinase 2 (MARK2), is a member of evolutionally conserved PAR1/MARK serine/threonine kinase family, which plays a key role in the establishment and maintenance of cell polarity at least partly by phosphorylating microtubule-associated proteins (MAPs) that regulate microtubule stability. PAR1b has also been reported to influence actin cytoskeletal organization, raising the possibility that PAR1b functionally interacts with the Rho family of small GTPases, central regulators of the actin cytoskeletal system. Consistent with this notion, PAR1 was recently found to be physically associated with a RhoA-specific guanine nucleotide exchange factor H1 (GEF-H1). This observation suggests a functional link between PAR1b and GEF-H1. Here we show that PAR1b induces phosphorylation of GEF-H1 on serine 885 and serine 959. We also show that PAR1b-induced serine 885/serine 959 phosphorylation inhibits RhoA-specific GEF activity of GEF-H1. As a consequence, GEF-H1 phosphorylated on both of the serine residues loses the ability to stimulate RhoA and thereby fails to induce RhoA-dependent stress fiber formation. These findings indicate that PAR1b not only regulates microtubule stability through phosphorylation of MAPs but also influences actin stress fiber formation by inducing GEF-H1 phosphorylation. The dual function of PAR1b in the microtubule-based cytoskeletal system and the actin-based cytoskeletal system in the coordinated regulation of cell polarity, cell morphology, and cell movement.
Highlights
Polarity-regulating kinase Partitioning-defective 1b (PAR1b) is involved in regulation of the actin cytoskeleton
guanine nucleotide exchange factor H1 (GEF-H1) phosphorylated on both of the serine residues loses the ability to stimulate RhoA and thereby fails to induce RhoA-dependent stress fiber formation. These findings indicate that PAR1b regulates microtubule stability through phosphorylation of microtubule-associated proteins (MAPs) and influences actin stress fiber formation by inducing guanine nucleotide exchange factors (GEFs)-H1 phosphorylation
This increase in stress fiber formation by PAR1b-specific siRNA was reverted by ectopic expression of PAR1b, which is encoded by an siRNA-resistant PAR1b cDNA (Fig. 1, A/C), or by treatment of cells with Y-27632, a chemical reagent that inhibits ROCK, the downstream target of RhoA (Fig. 1D and supplemental Fig. S3)
Summary
Polarity-regulating kinase PAR1b is involved in regulation of the actin cytoskeleton. PAR1b has been reported to influence actin cytoskeletal organization, raising the possibility that PAR1b functionally interacts with the Rho family of small GTPases, central regulators of the actin cytoskeletal system Consistent with this notion, PAR1 was recently found to be physically associated with a RhoA-specific guanine nucleotide exchange factor H1 (GEF-H1). Phosphorylation-dependent regulation of microtubule stability by PAR1 is thought to underlie the asymmetric distribution of molecules that mediates the establishment and maintenance of epithelial cell polarity [4, 5, 7, 8]. Upon ectopic expression of PAR1b, cells display a marked decrease in cortical actin [16] This observation raises the possibility that PAR1b modulates the function of RhoA, which is positively regulated by guanine nucleotide exchange factors (GEFs) and negatively regulated by GTPase-activation proteins (GAPs) [17, 18]. We obtained evidence supporting the idea that PAR1b influences RhoA-mediated regulation of the actin cytoskeletal system via GEF-H1 phosphorylation
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