Science Spotlight

Binding of an ubiquitin ligase complex stabilizes focal adhesions

Cas is phosphorylated specifically in FAs at the front of the cell. SOCS6 binds to pYCas in the FAs and locally inhibits pYCas via CRL5 and the proteasome. In cells lacking SOCS6 or Cul5, or in which SOCS6 cannot access FAs or cullins or the proteasome is inhibited, then pYCas stimulates FA disassembly, dependent on microtubules.
Image provided by Dr. Teckchandani

Cell migration is an important process during embryonic development, wound healing, and cancer cell invasion.  When cells move, they make structures called focal adhesions that contact the surrounding surface.  Focal adhesions are made up of a dense network of proteins, including transmembrane proteins, which contact proteins on the inside and outside of the cell.  Contact with proteins on the outside of the cell engages focal adhesion proteins that interact with dynamic actin fibers inside the cell.  Actin fibers grow toward the focal adhesions and push against the membrane, propelling the cell forward.  This force leads to changes in the structure of key proteins in the focal adhesion that then recruit more proteins to build up the focal adhesion, forming a positive feedback loop to promote focal adhesion assembly. In addition to this mechanically driven feedback circuit, localized molecular signaling pathways also regulate focal adhesion dynamics. Focal adhesion proteins are phosphorylated by focal adhesion kinase (FAK) and Src family kinases (SFKs). The focal adhesion protein Cas is phosphorylated by Src family kinases when other proteins in the focal adhesion (FA) come into contact with extracellular proteins.  Researchers have also uncovered that mechanically stretching Cas enhances its phosphorylation by SFKs.  Paradoxically, however, SFK signaling and phosphorylated Cas stimulate disassembly of focal adhesions. How focal adhesions at the leading edge maintain a productive balance between assembly and disassembly to push the cell forward remains unclear.  Postdoctoral fellow Dr. Anjali Teckchandani in Dr. Jonathan Cooper’s Laboratory (Basic Sciences Division) recently published an investigation in eLife reporting that phosphorylated Cas is prevented from promoting disassembly of focal adhesions at the leading edge through localized inhibition by a protein complex called CRLSOCS6
In the study, the scientists tracked the dynamics of individual fluorescently labeled focal adhesions using TIRF microscopy.  In control migrating MCF10A cells, a non-tumorigenic breast epithelial cell line, they observed large FAs at the leading edge of the cell.  The scientists’ previous work implicated Cullin 5, a protein in the CRL (cullin RING ubiquitin E3 ligase) complex, in the migration of breast epithelial cells.  When they knocked down expression of Cullin5, the large FAs were absent.  To measure dynamics, they recorded movies of moving cells and measured rates of assembly and disassembly for each FA.  They measured no difference in the average rate of FA assembly or disassembly at the front versus the back of an untreated migrating cell.  When they knocked down expression of Cullin5, they found that both the average rate of assembly and disassembly increased by two-fold, at the leading edge but not the back of the cell.
After determining that Cullin5 did not appear enriched in FAs compared to the overall cytosol of the cell, the scientists instead tracked the CRL complex substrate receptors, the SOCS proteins.  They detected an enrichment of SOCS6 at FAs that was lost upon siRNA knockdown of Cas and also when SFKs were inhibited with a drug.  Furthermore, a point mutation in the Cas SH2 domain prevented SOCS6 localization at FAs.  These data suggest that SOCS6 binds to SFK phosphorylated Cas in FAs at the leading edge.

Knockdown of Cullin5 and SOCS6 led to quicker disassembly of FAs following treatment and washout of the drug nocodazole, which stabilizes FAs by inhibiting microtubule stability. This suggested that Cul5 and SOCS6 stabilize FAs. To test whether the localization of SOCS6 at FAs was required for this effect, the scientists artificially localized SOCS6 to another location in the cell, the mitochondria, and measured FA turnover following nocodazole washout.  They made use of an optogenetic technique where two proteins with special modifications can be stimulated to bind one another in the presence of blue light. They introduced the photosensitive modifications on SOCS6 and a mitochondrial membrane protein. When the modified SOCS6 and mitochondrial protein were expressed in cells with unmodified SOCS6 expression knocked down by siRNA, the scientists found that cells kept in the dark had slow FA disassembly at a rate comparable to untreated cells.  When the same cells were kept in the light to stimulate the localization of SOCS6 to the mitochondria, the rate of FA disassembly mirrored knockdown of SOCS6 alone. These results and others suggest that SOCS6 must bind to phosphorylated Cas in FAs at the leading edge of cells to inhibit FA disassembly. 

Dr. Cooper explains that their results "suggest that Cas is modified with ubiquitin, and possibly degraded, while it is in or very close to the leading edge focal adhesion sites, slowing disassembly. The regulated binding of CRL5SOCS6 to Cas may locally inhibit FA disassembly and allow adhesions to grow at the leading edge." "New approaches will be required to measure the exact sequence of events – Cas binding, phosphorylation, SOCS6 binding, ubiquitination and proteolysis – during cell migration," said Cooper. 

Teckchandani A, Cooper JA.  2016.  "The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells."  eLife.  10.7554/eLife.17440

This work was supported by the National Institutes of Health.

See also the Fred Hutch News article covering this work: