A non-mitotic role for Aurora kinase A as a direct activator of cell migration upon interaction with PLD, FAK and Src.

Timely activation of Aurora kinase A (AURA, also known as AURKA) is vital for centrosome formation and the progression of mitosis. Nonetheless, it is still unclear if and when other cellular functions are activated by AURA. We report here that Src phosphorylates and activates AURA at T288, and AURA also activates focal adhesion kinase (FAK, also known as PTK2), leading to initiation of cell movement. An additional and new way by which AURA is regulated, is by phospholipase D2 (PLD2), which causes AURA activation. In addition, AURA phosphorylates PLD, so both proteins engage in a positive reinforcement loop. AURA and PLD2 form a protein­protein complex and colocalize to cytoplasmic regions in cells. The reason why PLD activates AURA is because of the production of phosphatidic acid by the lipase, which binds directly to AURA, with the region E171­E211 projected to be a phosphatidic-acid-binding pocket. Furthermore, this direct interaction with phosphatidic acid enhances tubulin polymerization and cooperates synergistically with AURA, FAK and Src in yielding a fully effectual cellular migration. Thus, Src and FAK, and PLD and phosphatidic acid are new upstream regulators of AURA that mediate its role in the non-mitotic cellular function of cell migration.

The molecular basis of leukocyte adhesion involving phosphatidic acid and phospholipase D.

Defining how leukocytes adhere to solid surfaces, such as capillary beds, and the subsequent migration through the extracellular matrix, is a central biological issue. We show here that phospholipase D (PLD) and its enzymatic reaction product, phosphatidic acid (PA), regulate cell adhesion of immune cells (macrophages and neutrophils) to collagen and have defined the underlying molecular mechanism in a spatio-temporal manner that coincides with PLD activity timing. A rapid (t½ = 4 min) and transient activation of the PLD1 isoform occurs upon adhesion, and a slower (t½ = 7.5 min) but prolonged (>30 min) activation occurs for PLD2. Importantly, PA directly binds to actin-related protein 3 (Arp3) at EC50 = 22 nm, whereas control phosphatidylcholine did not bind. PA-activated Arp3 hastens actin nucleation with a kinetics of t½ = 3 min at 300 nm (compared with controls of no PA, t½ = 5 min). Thus, PLD and PA are intrinsic components of cell adhesion, which reinforce each other in a positive feedback loop and react from cues from their respective solid substrates. In nascent adhesion, PLD1 is key, whereas a sustained adhesion in mature or established focal points is dependent upon PLD2, PA, and Arp3. A prolonged adhesion could effectively counteract the reversible intrinsic nature of this cellular process and constitute a key player in chronic inflammation.

Macrophage migration arrest due to a winning balance of Rac2/Sp1 repression over ß-catenin-induced PLD expression.

Monocytes and neutrophils infiltrate into tissues during inflammation and stay for extended periods of time until the initial insult is resolved or sometimes remain even longer in the case of chronic inflammation. The mechanism as to why phagocytes become immobilized after the initial cell migration event is not understood completely. Here, we show that overexpression or hyperactivation of Rac2 decreases sustained chemotactic responses of macrophages to MCP-1/CCL2. The resulting leukocyte arrest is not caused by a diminished availability of the cytokine receptor CCR2 that remains intact during MCP-1 stimulation. We show a novel mechanism that links the Rac2-dependent arrest of chemotaxis to decreased expression of PLD2 through the transcription regulator Sp1. Prolonged Rac2 activity leads to nuclear overactivation of Sp1, which acts as a repressor for PLD2. Also, another signaling component plays a regulatory role: ß-catenin. Although early times of stimulation (≈ 20 min) with MCP-1/CCL2 resulted in activation of ß-catenin with a positive effect on PLD2, after ≈ 3 h of stimulation, the levels of ß-catenin were reduced and not able to prevent the negative effect of Rac2 on PLD2 activity. This is a novel molecular mechanism underlying immobilization of monocyte/macrophage migration that is important for the physiological maintenance of leukocytes at the site of inflammation. If this immobilization is prolonged enough, it could lead to chronic inflammation.