Accumulating evidence in various systems converges on the idea that multiple inputs regulate protein activity and subcellular localization, a concept referred to as coincidence detection. The regulation of actin filament nucleation may also follow this scheme. For example, N-WASP, an autoinhibited regulator of the Arp2/3 complex, can be cooperatively activated and recruited to the membrane by interactions with Cdc42 through its GBD, phosphoinositides through its adjacent BD (Prehoda et al.
), and SH3 domain-containing proteins through its proline-rich region (Takenawa and Suetsugu, 2007
; Derivery and Gautreau, 2010
The regulation of mDia formins resembles that of N-WASP, as they are also autoinhibited proteins regulated by small GTPases cooperating with other coactivators (Seth et al.
; Dominguez, 2010
). Protein interaction with phospholipids at the membrane frequently plays a role in coincidence detection, but its importance is poorly understood for mDia formins, and in particular for mDia2. Here, we found that interactions of mDia2 with GTPases and phospholipids contribute to its localization at the plasma membrane in an actin-independent manner and that this activity is mediated by the coordinated actions of several N-terminal domains in a previously unappreciated manner.
Our main finding is that the BD plays an essential role in plasma membrane targeting of the Nt of mDia2. Its effect appears to be specific, as the C-terminal basic stretch adjacent to DAD (Wallar et al.
) is insufficient to recruit the Ct of mDia2 to the plasma membrane. Interestingly, a computer algorithm that searches for potential unstructured membrane-binding sites in protein sequences (Brzeska et al.
) also identifies the BD of mDia2 as a potential membrane-binding site.
Protein–lipid interactions commonly rely on electrostatic forces and hydrophobic interactions, consistent with the amphipathic nature of membrane phospholipids. Electrostatic interactions likely contribute to the association of the BD with the plasma membrane in vivo, first, because the deletion of the highly basic stretch within the first 37 amino acids of the BD significantly impairs membrane targeting of mDia2 fragments and, second, because the BD binds to acidic phospholipids in vitro. The apparent broad specificity of the BD for acidic phospholipids is reminiscent of the regulation of the WAVE complex, an activator of the Arp2/3 complex, by a range of charged acidic phospholipids (Lebensohn and Kirschner, 2009
). The remainder of the BD contains two clusters of hydrophobic amino acids, predicted to form amphipathic helices. Deletion of these clusters decreases membrane targeting, suggesting that they also contribute to plasma membrane binding. These putative helices could promote membrane binding by several nonexclusive mechanisms: formation of a single folding unit with the GBD for GTPase binding; insertion into the membrane bilayer, as has been suggested for the BD of mDia1 (Ramalingam et al.
); clustering of basic amino acids into a common membrane-binding interface; or interaction with some membrane-associated proteins.
Although the BD is important for membrane targeting, it is not solely responsible for the strong binding of the mDia2 Nt. Generally, proteins activated by small GTPases are thought to be also recruited to the membrane by these GTPases. Indeed, RhoA-dependent recruitment of mDia2 to the cytokinetic ring has been recently demonstrated (Watanabe et al.
). The identity of the small GTPase targeting mDia2 to the plasma membrane in interphase remained unclear, however. Among several GTPases reported to interact with mDia2 (Alberts et al.
; Peng et al
; Pellegrin and Mellor
; Wallar et al.
; Ji et al
; Lammers et al.
, only Rif potently and specifically enhanced the membrane localization of mDia2 constructs, suggesting that RhoA, Cdc42, and Rac1 may target mDia2 to other subcellular locations. Indeed, we have observed that overexpressed Rif is much more enriched at the membrane than are other GTPases, all of which are believed to interact with the membrane through prenylation of their C-terminal CAAX motifs. Thus, additional mechanisms may be involved in enhancing the membrane localization of Rif.
The structural basis for the interaction of mDia formins with GTPases is best known for the mDia1–RhoC complex, the crystal structure of which has been determined (Otomo et al.
; Rose et al.
). It showed that both the G region and the DID of mDia1 make specific contacts with the GTPase. Similar contacts were observed for a complex of an mDia2-mimicking mutant of mDia1 and Cdc42 or Rac1 (Lammers et al.
), although the structure of the actual mDia2 with any GTPase has not yet been determined. The biochemical analysis of the mDia2-Rif interaction (Pellegrin and Mellor, 2005
) did not focus on whether both the G region and DID were required for binding. Here, we used purified proteins to demonstrate a direct interaction between active Rif and G-DID–containing constructs of mDia2, whereas the interaction of the fragment BD-G with was much weaker. Thus, both G and DID of mDia2 are needed for optimal binding to Rif, which is analogous to the interactions of mDia1 with Rho family GTPases. Despite the ability of the BD to enhance the plasma membrane localization of the N-terminal mDia2 constructs in a Rif-dependent manner in cells, however, the BD is not involved in direct interaction between the N terminus of mDia2 and Rif. These findings suggest that Rif indirectly enhances the recruitment of the BD to the plasma membrane in cells, for example, by changing the plasma membrane composition through other effectors or signaling pathways.
Although both G and DID participate in GTPase-dependent targeting of mDia2 to the plasma membrane, surprisingly, they are not sufficient, as the construct G-DID fails to localize appreciably to the plasma membrane even in Rif-expressing cells. The addition of DD-CC to G-DID, however, rescues plasma membrane targeting, possibly through dimerization, which allows for multivalent binding (increased avidity) of the BD-G-DID module. If this idea is correct, the dimerization may require both the DD and CC domains, as the removal of the CC from Nt severely decreases plasma membrane targeting. In mDia1, however, the DD is sufficient to mediate dimerization in vitro (Otomo et al.
) suggesting that the NtΔCC of mDia2 may also be a dimer. Another possibility is that the DD-CC–containing region has membrane-targeting capabilities of its own, as proposed for mDia1 (Copeland et al.
). Consistent with this idea, DID-DD-CC of mDia2 localizes to the cytokinetic ring by interacting with anillin (Watanabe et al.
). The N-terminal region of mDia2 containing partial DID, DD, and CC is also involved in the Abi1-dependent stabilization of mDia2 at filopodial tips (Yang et al.
). Inability of DD-CC or DID-DD-CC to accumulate at the plasma membrane, however, is not consistent with this possibility or with a scenario in which DD-CC–containing constructs dimerize with endogenous mDia2. Therefore, we currently favor an idea that the DD-CC module, in addition to dimerization, may cause a conformational change that allows for better binding of the GTPase by G-DID or improves binding of another target (for instance Abi1) by N-terminal domains. Consistent with this idea, it has been found recently that the N terminus of mDia1 correctly interacts with its C terminus only when the N terminus contains the CC domain (Nezami et al.
; Otomo et al.
Together, our results suggest a model for a mechanism of mDia2 targeting with implications for its activation (). We propose that the BD, which is expected to be accessible in the autoinhibited conformation of mDia2, mediates initial binding to the membrane through electrostatic, and possibly also hydrophobic, interactions. This initial binding allows mDia2 to linger at the plasma membrane until it encounters active Rif. Next, weak binding of Rif to the G region causes the displacement of the DAD from the DID, as proposed previously (Lammers et al.
), which would allow the GTPase to engage the DID to form a more stable complex at the membrane. The role of the DD-CC module in mDia2 is to dimerize and optimally arrange the N-terminal mDia2 domains for efficient Rif binding and/or for engagement of additional targeting molecules.
FIGURE 7: Model for plasma membrane targeting of mDia2. Step 1: Phospholipid binding. The initial targeting event occurs while mDia2 is still autoinhibited, yet its BD is accessible to bind acidic phospholipids of the plasma membrane through electrostatic interactions. (more ...)
Thus, mDia2 targeting, and possibly activation as well, occurs through extensive cooperation of all N-terminal domains, which together serve as a coincidence detection module recognizing at least two inputs: membrane phospholipids and a small GTPase. A similar mechanism may also be used to some extent by other mDia formins. Thus, mDia1 has a similarly charged, albeit slightly shorter, BD at the N terminus (Ramalingam et al.
), whereas the corresponding region of mDia3 has a slightly lower predicted pI of ~8, because it lacks the first cluster of basic amino acids. In contrast, other formins containing an N-terminal GBD (Schonichen and Geyer, 2010
) are not associated with an upstream basic sequence, suggesting that the BD-G module is specific for Diaphanous-related formins.