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Review
. 2014 Sep:182:73-9.
doi: 10.1016/j.chemphyslip.2014.01.002. Epub 2014 Jan 19.

Interplay between phosphoinositide lipids and calcium signals at the leading edge of chemotaxing ameboid cells

Joseph J Falke  1 , Brian P Ziemba  2
Affiliations
Review

Interplay between phosphoinositide lipids and calcium signals at the leading edge of chemotaxing ameboid cells

Joseph J Falke et al. Chem Phys Lipids. 2014 Sep.

Abstract

The chemotactic migration of eukaryotic ameboid cells up concentration gradients is among the most advanced forms of cellular behavior. Chemotaxis is controlled by a complex network of signaling proteins bound to specific lipids on the cytoplasmic surface of the plasma membrane at the front of the cell, or the leading edge. The central lipid players in this leading edge signaling pathway include the phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), both of which play multiple roles. The products of PI(4,5)P2 hydrolysis, diacylglycerol (DAG) and Ins(1,4,5)P3 (IP3), are also implicated as important players. Together, these leading edge phosphoinositides and their degradation products, in concert with a local Ca(2+) signal, control the recruitment and activities of many peripheral membrane proteins that are crucial to the leading edge signaling network. The present critical review summarizes the current molecular understanding of chemotactic signaling at the leading edge, including newly discovered roles of phosphoinositide lipids and Ca(2+), while highlighting key questions for future research.

Keywords: C2 domain; PDK1; PH domain; PI3K; PKC; Phosphatidylinositol lipid.

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Figures

Fig. 1
Fig. 1
Second messenger signals at the leading edge of polarized ameboid cells (Evans and Falke, 2007). Shown are polarized RAW macrophages illustrating (A) the ruffling leading edge in a phase contrast image, (B) the local Ca2+ signal revealed by YFP-PKCα recruited to the leading edge membrane in a wide field fluorescence image, and (C) the local PI(3,4,5)P3 signal revealed by GFP-PKB/AKT-PH domain recruited to the leading edge membrane in a wide field fluorescence image. (D) Schematic overview of parallel Ca2+ and PI(3,4,5)P3 (PIP3) signals that activate PH and C2 membrane targeting domains, triggering recruitment of signaling proteins to the leading edge membrane (Jin, 2013; Weiger and Parent, 2012; Cai and Devreotes, 2011; Kolsch et al., 2008; von Philipsborn and Bastmeyer, 2007; Evans and Falke, 2007; Corbalan-Garcia et al., 2007; Leonard and Hurley, 2011; Newton, 2009; Lemmon, 2008; Cho, 2001; Nalefski and Falke, 1996). The resulting bilayer-associated signaling pathway includes a positive feedback loop that controls actin and membrane remodeling essential for leading edge structure, stability, dynamics, and directed cell migration (Jin, 2013; Weiger and Parent, 2012; Cai and Devreotes, 2011; Hawkins et al., 2010; Swaney et al., 2010; Kolsch et al., 2008; Mortimer et al., 2008; von Philipsborn and Bastmeyer, 2007; Bourne and Weiner, 2002; Evans and Falke, 2007; Charest and Firtel, 2006).
Fig. 2
Fig. 2
Function of PH and C2 membrane targeting domains in the recruitment and activation of signaling proteins on target membrane surfaces (Corbalan-Garcia et al., 2007; Leonard and Hurley, 2011; Newton, 2009; Lemmon, 2008; Cho, 2001; Nalefski and Falke, 1996). First, a 2° messenger signal triggers the recruitment of the signaling protein targeting domain from the cytoplasm to the bilayer surface: many PH domains are recruited to the plasma membrane by a PI(3,4,5)P3 (PIP3) lipid signal, while the C2 domains of classical protein kinase C isoforms (cPKCs) are activated by Ca2+ binding and then dock to PS and PI(4,5)P2 (PIP2) on the plasma membrane. Second, the signaling domain of each recruited protein is activated by its proximity on the membrane surface to membrane-associated protein and/or lipid molecules that serve as effectors or substrates. At the leading edge membrane, PIP3 and Ca2+ both recruit signaling proteins and establish a signaling network on the bilayer surface. Lateral, 2-dimensional diffusion on the bilayer is required for the signaling proteins to encounter and bind their signaling partners, and for the resulting membrane-bound products to diffuse away. Thus, the lipid bilayer serves as a 2-dimensional platform for the assembly of the dynamic signaling circuit, and for the diffusion of its many components during signaling reactions.
Fig. 3
Fig. 3
Schematic, partial model of the intricate leading edge signaling circuit, illustrating known and hypothesized components and their interactions (Jin, 2013; Weiger and Parent, 2012; Cai and Devreotes, 2011; Hawkins et al., 2010; Collins and Meyer, 2009; Wei et al., 2012; Tsai and Meyer, 2012; Zamburlin et al., 2013; Henle et al., 2011; Swaney et al., 2010; Kolsch et al., 2008; Mortimer et al., 2008; von Philipsborn and Bastmeyer, 2007; Tian et al., 2010; Wei et al., 2009; Evans and Falke, 2007). Attractant signals switch on receptors and the Ras protein, which recruit and activate the lipid kinase activity of PI3K that phosphorylates the constitutive lipid PI(4,5)P2 (PIP2) to generate the signaling lipid PI(3,4,5)P3 (PIP3). Attractant signals may also activate plasma membrane Ca2+ channels that generate an essential Ca2+ influx at the leading edge. The PI(3,4,5)P3 and Ca2+ second messengers are essential players in the leading edge positive feedback loop (dashed line) along with one or more small GTPases of the Rho family, filamentous actin and PKCα. The PI(3,4,5)P3 signal recruits PDK1 to the leading edge where it is activated and stimulates branches leading to membrane and actin remodeling needed for leading edge expansion up the attractant gradient. The Ca2+ signal activates and recruits PKCα to the leading edge membrane where it is proposed to phosphorylate the PI(4,5)P2-sequestration region of MARCKS protein, which in turn is predicted to increase the free PI(4,5)P2 density and thereby activate PI3K and self-activate PKCα. Ca2+ may also activate PLC to generate a putative DAG lipid signal. Finally, PDK1 phosphorylation of PKCα in solution is essential for maturation and activation of the latter. On the membrane surface PDK1 and PKCα are hypothesized to combine and form a stable, inactive complex that could act as a brake to prevent runaway positive feedback. Proteins in boxes contain a PH domain (black box) or a C2 domain (white box). Second messengers are also highlighted (blue box).
Fig. 4
Fig. 4
Working model for the molecular interactions between the activities of PKCα, PI3K and PDK1 in the leading edge positive feedback loop. Shown are the three master kinases and their effector lipids on the cytoplasmic leaflet of the leading edge membrane. Kinase-active PKCα is bound via its Ca2+-occupied C2 domain to PS and PI(4,5)P2 (PIP2), and via its C1A and C1B domains to PS and DAG (Evans and Falke, 2007; Corbalan-Garcia et al., 2007; Corbin et al., 2007; Evans et al., 2006; Newton, 2009). This active PKCα is proposed to phosphorylate the PI(4,5)P2-sequestration region of MARCKS protein, thereby releasing PI(4,5)P2 from MARCKS and increasing the local free PI(4,5)P2 density at the leading edge (Golebiewska et al., 2008; McLaughlin et al., 2002; Wang et al., 2004; Gambhir et al., 2004). The newly released PIP2 helps counteract the loss of PI(4,5)P2 due to the enzymatic activities of PI3K and PLC (Jin, 2013; Wei et al., 2012; Tsai and Meyer, 2012; Vadas et al., 2011; Wei et al., 2009; Evans and Falke, 2007), and this PI(4,5)P2 release activates both PI3K that requires PI(4,5)P2 as a substrate, and PKCα that requires PI(4,5)P2 for stable plasma membrane association. PI3K converts PI(4,5)P2 to the signaling lipid PI(3,4,5)P3 (PIP3), which recruits many signaling proteins including PDK1 to the leading edge membrane. PDK1 is an inactive dimer that is converted on the membrane surface to an active monomer (Ziemba et al., 2013; Masters et al., 2010). The resulting PDK1 monomer may form an inhibitory complex with PKCα that has been isolated from cytoplasm but not yet investigated on a membrane surface (Le Good et al., 1998). Further studies are needed to test the proposed interactions between the three master kinases, and the roles of these interactions in leading edge positive feedback and signal amplification.
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