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Invasion by actin-driven membrane protrusions: Cortactin in focus

Cortactin’s multiple signaling domains

The actin binding protein cortactin plays an important role in several cellular functions involving plasma membrane changes that are dependent on a dendritic (i.e., branched) actin network: cell motility employing lamellipodia, clathrin dependent and independent endocytosis, host-pathogen interactions, maintenance of endothelial barrier integrity, and invadopodia-mediated cell invasion1.  Cortactin is a monomeric ~80 kDa protein that derives its name from its intracellular colocalization with cortical actin at the periphery of the cell2.  The amino terminus of cortactin harbors a domain rich in acidic amino acids (N-terminal acidic domain; NTA) that interacts with the seven subunit Arp2/3 complex that promotes dynamic actin filament branching3.  Cortactin’s NTA domain contains a DDW (Asp-Asp-Trp) motif that has been observed in the Arp2/3 binding regions of other known actin nucleation promoting factors (NPFs) as either DDW or DEW (e.g., WASP, N-WASP, Myo3, ActA)4 (Fig. 1).  NPFs are divided into two classes based on their role in promoting Arp2/3-mediated filamentous actin (F-actin) branching5.   Class I NPFs like those mentioned above have a primary role in promoting Arp2/3 activation to drive filament branching, whereas Class II NPFs like cortactin function in branch formation and stabilization of the dynamic branched actin assembly6,7.  Notably, while cortactin can directly activate Arp2/3 mediated F-actin branching, this activity is much weaker than is observed for Class I NPFs6.  This is due in part to cortactin lacking the monomeric globular actin (G-actin) binding domain of class I NPFs5.  Consistent with cortactin’s role in stabilizing branched filaments, it has a central domain with 6.5 repeats of a 37-residue long sequence that binds F-actin2,4 (Fig. 1).  This domain is followed by a helical region and a proline-rich region, containing multiple sites of post-translational modifications (PTMs), and an SH3 domain that is used to recruit other proteins, including the Class I NPF N-WASP to the Arp2/3 complex at the filament branch point (for a list of binding partners, see ref. 8).  

 

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cortactin-primary-structure

Figure 1: Schematic diagram of cortactin’s primary structure with key post-translational modifications and binding domains highlighted.

 

The association of cortactin with aggressive cancers

The F-actin rich cellular protrusions known as invadopodia were originally identified in human cancer cell lines and were named for their invasive nature9.  Invadopodia are 0.8-1 µm diameter membrane extensions that are 2-5 µm in length and are found on basal membranes that face the extracellular matrix (ECM)10.  Cortactin is essential for invadopodia formation and for their focused delivery of matrix metalloproteases to promote ECM degradation and promote cellular invasion into neighboring tissues10-12.  Several human cancers have been shown to exhibit elevated expression of cortactin including breast, colorectal, ovarian, hepatic, gastric, esophageal, melanomas, and glioblastomas8,13.  Consequently, cortactin has become an important biomarker for malignant metastatic cancers and its elevated expression is frequently associated with a poor patient prognosis.

 

The importance of cortactin post-translational modifications

The regulation of cortactin’s activity through PTMs is very complex and growing evidence points to a high degree of cross-talk between different PTMs (Fig. 1).  Phosphorylation of four serine residues (S405, S418, S298 and S348) by one or more kinases (ERK, PAK1, or PKD1) regulates cortactin’s activity8. ERK phosphorylation on S405/S418 in the proline-rich domain of cortactin is thought to induce a conformational change that exposes the SH3 domain, allowing the binding and activation of class I NPFs N-WASP or WASP14.  In contrast, phosphorylation of tyrosine residues Y421, Y466, and Y482 by Src family kinases, and presumably other tyrosine kinases that target these residues15,16, results in the loss of cortactin’s ability to promote Arp2/3-mediated F-actin branching either directly or through indirect activation and recruitment of N-WASP or WASP14,17.  This reciprocal relationship between serine and tyrosine phosphorylation has been termed the “S-Y switch”18.  It is interesting that both serine and tyrosine phosphorylation of cortactin act as signals for its degradation.  ERK phosphorylation of the aforementioned serines acts as a trigger for K79 ubiquitination through cortactin interactions with the β-Trcp subunit of the E3 ligase complex, which ultimately leads to its proteosomal degradation19.  In a similar manner, tyrosine phosphorylation of cortactin is a signal for its cleavage by the calcium-dependent protease calpain20.  Cortactin’s ability to interact with F-actin, and consequently activate Arp2/3, is also modulated by lysine acetylation/deacetylation mediated in part by PCAF and HDAC6, respectively21.  Interestingly, recent evidence suggests a competition exists between tyrosine phosphorylation and lysine acetylation even though both PTMs negatively affect the activity of cortactin22.  This alludes to yet another layer of complexity in the cross-talk of cortactin PTMs and it will be exciting to see how the details of these interconnected signaling pathways converge to regulate cortactin at the actin filament branchpoint.

 

As new actin research tools, including those to study the PTMs of actin and its associated proteins, become available, the physiological functions of cortactin will be further elucidated using purified actin and actin binding proteins from Cytoskeleton, Inc.

 

Actin Related Research Tools

  ProteinSource   Purity   Cat. #    Amount
 

Actin Protein

Rabbit skeletal muscle >99% 

AKL99-A
AKL99-B

 

4 x 250 µg
2 x 1 mg

 

Actin Protein

Porcine Brain >99% 

APHL99-A
APHL99-B

 

2 x 250 µg
1 x 1 mg

 

Pre-formed Actin Filaments

Rabbit skeletal muscle >99% 

AKF99-A
AKF99-B

 

1 x 1 mg
5 x 1 mg

 Pyrene Actin ProteinRabbit skeletal muscle>99%AP05-A
AP05-B		1 x 1 mg
5 x 1 mg
 Biotinylated Actin ProteinRabbit skeletal muscle 

>99%

 

AB07-A
AB07-B

 

5 x 20 mg
20 x 20 mg

         
 Kit    Cat. # Amount
 

G-actin/F-actin In Vivo Biochem Kit™
 

    		BK037 30-100 assays
 

Actin Binding Protein Spin-down Assay Biochem Kit™
 

    		BK013 30-100 assays
 

Actin Polymerization Biochem Kit™

    		BK003 24-30 assays

 

References

  1. Cosen-Binker LI and Kapus A. 2006. Cortactin: The gray eminence of the cytoskeleton. Physiology. 21: 352-361.
  2. Wu H and Parsons JT. 1993. Cortactin, an 80/85-Kilodalton pp60 src substrate, is a filamentous actin-binding protein enriched in the cell cortex. J. Cell Biol. 120: 1417-1426.
  3. Mullins RD et al. 1998. The interaction of the Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of actin filaments. Proc. Natl. Acad. Sci. USA. 95: 6181-6186.
  4. Weed SA et al. 2000. Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex. J. Cell Biol. 151:29-40.
  5. Goley ED and Welch MD. 2006. The Arp2/3 complex: an actin nucleator comes of age. Nat. Rev. Mol. Cell Biol. 7: 713-726.
  6. Weaver AM et al. 2001. Cortactin promotes and stabilizes Arp2/3-induced actin filament network formation. Curr. Biol. 11: 370-374.
  7. Egile C et al. 2005. Mechanism of filament nucleation and branch stability revealed by the structure of the Arp2/3 complex at actin branch junctions. PLoS Biol. 3: 1902-1909.
  8. Kirkbride KC et al. 2011. Cortactin a multifunctional regulator of cellular invasiveness.  Cell Adh. Migr. 5: 187-198.
  9. Chen WT. 1989. Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells. J. Exp. Zool. 251: 167-185.
  10. Artym VV et al. 2006. Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase in invadopodia: defining the stages of invadopodia formation and function. Cancer Res. 66: 3034-3043.
  11. Clark ES and Weaver AM. 2008. A new role for cortactin in invadopodia: Regulation of protease secretion. Eur. J. Cell Biol. 87: 581-590.
  12. Clark ES et al. 2007. Cortactin is an essential regulator of matrix metalloprotease secretion and extracellular matrix degradation in invadopodia. Cancer Res. 67: 4227-4235. 
  13. MacGrath SM and Koleske AJ. 2012. Cortactin in cell migration and cancer at a glance. J. Cell Sci. 125: 1621-1626.
  14. Martinez-Quiles N et al. 2004. Erk/Src phosphorylation of cortactin acts as a switch on-switch off mechanism that controls its ability to activate N-WASP. Mol. Cell Biol. 24: 5269-5280.
  15. Sangar W et al. 2007. Fer-mediated cortactin phosphorylation is associated with efficient fibroblast migration and is and is dependent on reactive oxygen species generation during integrin-mediated cell adhesion. Mol. Cell Biol. 27: 6140-6152.
  16. Boyle SN et al. 2007. A critical role for cortactin phosphorylation by Abl-family kinases in PDGF-induced dorsal-wave formation. Curr Biol. 17: 445-451.
  17. Huang C et al. 1997. Down-regulation of the filamentous actin cross-linking activity of cortactin by Src-mediated tyrosine phosphorylation. J. Biol. Chem. 272: 13911-13915.
  18. Lua BL and Low BC. 2005. Cortactin phosphorylation as a switch for actin cytoskeleton network and cell dynamics control. FEBS Lett. 579: 577-585.
  19. Zhao J et al. 2012. Extracellular signal-regulating kinase (ERK) regulates cortactin ubiquitination and degradation in lung epithelial cells. J. Biol. Chem. 287: 19105-19114.
  20. Huang C et al. 1997. Proteolysis of platelet cortactin by calpain. J. Biol. Chem. 272: 19248-19252.
  21. Zhang X et al. 2007. HDAC6 modulates cell motility by altering the acetylation level of cortactin. Mol. Cell. 27: 197-213.
  22. Meiler E et al. 2012. Cortactin tyrosine phosphorylation promotes its deacetylation and inhibits cell spreading. PLoS ONE. 7: e33662.