A tripartite complex containing MRCK modulates lamellar actomyosin retrograde flow.

Actomyosin retrograde flow underlies the contraction essential for cell motility. Retrograde flow in both lamellipodia and lamella is required for membrane protrusion and for force generation by coupling to cell adhesion. We report that the Rac/Cdc42-binding kinase MRCK and myosin II-related MYO18A linked by the adaptor protein LRAP35a form a functional tripartite complex, which is responsible for the assembly of lamellar actomyosin bundles and of a subnuclear actomyosin network. LRAP35a binds independently to MYO18A and MRCK. This binding leads to MRCK activation and its phosphorylation of MYO18A, independently of ROK and MLCK. The MRCK complex moves in concert with the retrograde flow of actomyosin bundles, with MRCK being able to influence other flow components such as MYO2A. The promotion of persistent protrusive activity and inhibition of cell motility by the respective expression of wild-type and dominant-negative mutant components of the MRCK complex show it to be crucial to cell protrusion and migration.

Arg kinase-binding protein 2 (ArgBP2) interaction with α-actinin and actin stress fibers inhibits cell migration.

Cell migration requires dynamic remodeling of the actomyosin network. We report here that an adapter protein, ArgBP2, is a component of α-actinin containing stress fibers and inhibits migration. ArgBP2 is undetectable in many commonly studied cancer-derived cell lines. COS-7 and HeLa cells express ArgBP2 (by Western analysis), but expression was detectable only in approximately half the cells by immunofluorescence. Short term clonal analysis demonstrated 0.2-0.3% of cells switch ArgBP2 expression (on or off) per cell division. ArgBP2 can have a fundamental impact on the actomyosin network: ArgBP2 positive COS-7 cells, for example, are clearly distinguishable by their denser actomyosin (stress fiber) network. ArgBP2γ binding to α-actinin appears to underlie its ability to localize to stress fibers and decrease cell migration. We map a small α-actinin binding region in ArgBP2 (residues 192-228) that is essential for these effects. Protein kinase A phosphorylation of ArgBP2γ at neighboring Ser-259 and consequent 14-3-3 binding blocks its interaction with α-actinin. ArgBP2 is known to be down-regulated in some aggressively metastatic cancers. Our work provides a biochemical explanation for the anti-migratory effect of ArgBP2.

Dynamic instability of clathrin assembly provides proofreading control for endocytosis.

Clathrin-mediated endocytosis depends on the formation of functional clathrin-coated pits that recruit cargos and mediate the uptake of those cargos into the cell. However, it remains unclear whether the cargos in the growing clathrin-coated pits are actively monitored by the coat assembly machinery. Using a cell-free reconstitution system, we report that clathrin coat formation and cargo sorting can be uncoupled, indicating that a checkpoint is required for functional cargo incorporation. We demonstrate that the ATPase Hsc70 and a dynamic exchange of clathrin during assembly are required for this checkpoint. In the absence of Hsc70 function, clathrin assembles into pits but fails to enrich cargo. Using single-molecule imaging, we further show that uncoating takes place throughout the lifetime of the growing clathrin-coated pits. Our results suggest that the dynamic exchange of clathrin, at the cost of the reduced overall assembly rates, primarily serves as a proofreading mechanism for quality control of endocytosis.

Real-Time Monitoring of Clathrin Assembly Kinetics in a Reconstituted System.

Clathrin-coated pits (ccp) are important structures that cells use for internalizing materials and regulating plasma membrane homeostasis. We had previously described an assay of reconstituting ccp assembly on sheets of basal plasma membranes. Here, we describe a workflow to adapt this system for monitoring the assembly of ccps over time using total internal reflection fluorescence (TIRF) microscopy.

ArhGAP15, a novel human RacGAP protein with GTPase binding property.

We have previously described a partial cDNA sequence encoding a RhoGAP protein, GAP25 that is homologous to the recently reported ArhGAP9 and ArhGAP12. We now describe a related new member ArhGAP15 that shares a number of domain similarities, including a pleckstrin homology (PH) domain, a RhoGAP domain and a novel motif N-terminal to the GAP domain. This novel motif was found to be responsible for nucleotide-independent Rac1 binding. Using swop mutants of Rac/Cdc42, we have established that the binding is through the C-terminal half of Rac1. The GAP domain of ArhGAP15 showed specificity towards Rac1 in vitro. The PH domain is required for ArhGAP15 to localize to cell periphery and over-expression of the full-length ArhGAP15, but not the mutant with a partial deletion of the PH domain, resulted in an increase in actin stress fibers and cell contraction. These morphological effects can be attenuated by the co-expression of dominant negative Rac1(N17). HeLa cells expressing ArhGAP15 were also resistant to phorbol myristatate acetate treatment, suggesting that ArhGAP15 is a potential regulator of Rac1.

Chelerythrine perturbs lamellar actomyosin filaments by selective inhibition of myotonic dystrophy kinase-related Cdc42-binding kinase.

Cell movement requires forces generated by non-muscle myosin II (NM II) for coordinated protrusion and retraction. The Cdc42/Rac effector MRCK regulates a specific actomyosin network in the lamella essential for cell protrusion and migration. Together with the Rho effector ROK required for cell rear retraction, they cooperatively regulate cell motility and tumour cell invasion. Despite the increasing importance of ROK inhibitors for both experimental and clinical purposes, there is a lack of specific inhibitors for other related kinases such as MRCK. Here, we report the identification of chelerythrine chloride as a specific MRCK inhibitor. Its ability to block cellular activity of MRCK resulted in the specific loss of NM II-associated MLC phosphorylation in the lamella, and the consequential suppression of cell migration.

Phosphorylation of myosin phosphatase targeting subunit 3 (MYPT3) and regulation of protein phosphatase 1 by protein kinase A.

Myosin phosphatase targeting subunit 3 (MYPT3) and transforming growth factor-beta-inhibited membrane-associated protein (TIMAP) are two closely related myosin-binding targeting subunits of protein phosphatase 1 (PP1c) with a characteristic CAAX (where AA indicates aliphatic amino acid) box at the C termini. Here we show that MYPT3 can be a substrate for protein kinase A (PKA). We first mapped the multiple phosphorylation sites within a central conserved motif. Deletion or mutations of this motif resulted in enhancement of the associated PP1c activity, suggesting that phosphorylation of MYPT3 may play an important role in regulating PP1c catalytic activity. However, unlike the other known MYPTs, which upon phosphorylation inhibit PP1c, PKA phosphorylation of MYPT3 resulted in PP1c activation, indicating a different mode of action. There is a direct interaction between the central conserved phosphorylated site motif with the N-terminal ankyrin repeat region; this interaction was significantly reduced with MYPT3 phosphorylation or acidic phosphorylation site mutations, with concomitant alterations in biochemical and morphological consequences. We therefore propose a novel mechanism for the phosphorylation of MYPT3 by PKA and activation of the catalytic activity through direct interaction of a central region of MYPT3 with its N-terminal region.