PI(3,4,5)P ₃ and PI(4,5)P ₂ Lipids Target Proteins with Polybasic Clusters to the Plasma Membrane

Abstract

Many signaling, cytoskeletal, and transport proteins have to be localized to the plasma membrane (PM) in order to carry out their function. We surveyed PM-targeting mechanisms by imaging the subcellular localization of 125 fluorescent protein-conjugated Ras, Rab, Arf, and Rho proteins. Out of 48 proteins that were PM-localized, 37 contained clusters of positively charged amino acids. To test whether these polybasic clusters bind negatively charged phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] lipids, we developed a chemical phosphatase activation method to deplete PM PI(4,5)P 2 . Unexpectedly, proteins with polybasic clusters dissociated from the PM only when both PI(4,5)P 2 and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] were depleted, arguing that both lipid second messengers jointly regulate PM targeting.Small guanosine triphosphatases (GTPases) from the Ras, Rho, Arf, and Rab subfamilies often exert their role at the PM where they control diverse signaling, cytoskeletal, and transport processes (1-3). KRas, CDC42, and other family members require a cluster of positively charged amino acids for PM localization and activity (2, 4). In vitro studies indicate that the physiological PM binding partner of such polybasic clusters could be phosphatidylserine, which has one negative charge, or the less abundant lipid second messenger PI(4,5)P 2 , which has four negative charges (5-7). We took a genomic survey approach and investigated PM-targeting mechanisms by confocal imaging of 125 cyan fluorescent protein (CFP)-tagged constitutively active small GTPases (8). Expression in NIH3T3 and HeLa cells showed that 48 small GTPases were fully or partially localized to the PM (Fig. 1A and fig. S1).Thirty-seven of these PM-localized small GTPases had C-terminal polybasic clusters consisting of four or more Lys or Arg residues at positions 5 to 20 from the C terminus ( Fig. 1B and fig. S1). Polybasic clusters were found in three forms: They were present together with N-terminal myristoylation consensus sequences (as in Arl4) (9) or with C-terminal prenylation consensus sequences (as in KRas) (5, 6, 10), or they lacked lipid modifications (as in Rit) (11). We called these three combinations polybasic-myristoyl, polybasic-prenyl, and polybasic-nonlipid PM-targeting motifs, respectively. A number of remaining PMtargeted small GTPases had a combined prenylation and palmitoylation consensus sequence that mediated PM targeting without requiring polybasic amino acids (as does that of HRas) (Fig. 1D).To test whether polybasic clusters are anchored to the PM by binding to PI(4,5)P 2 (14), we hydrolyzed PM PI(4,5)P 2 by rapid targeting of Inp54p, a 5′ specific PI(4,5)P 2 phosphatase (15), to the PM. This method is based on a PM-localized FK506-binding protein (FKBP12)-rapamycin-binding (FRB) construct and a cytosolic Inp54p enzyme conjugated with FKBP12 (CF-Inp) that can be translocated to the PM by chemical heterodimerization by using a rapamycin analog, iRap (16).In experiments where we monitored PI(4,5)P...

Oncogenic signaling of class I PI3K isoforms

“…Sequence alignment of the class I PI3K isoforms reveals many basic residues, mostly lysines, within the C2 domain. Basic residues are known to be involved in lipid-protein interactions (Heo et al, 2006). An area of concentrated basic residues in the C2 domain of p110d, containing three lysines, that is, K402, K409 and K410, is not observed in any other class I isoform.…”

“…The C2 domain in p110g has also been shown to mediate recruitment to membranes (Walker et al, 2000). Positively charged amino acids are critically involved in membrane binding (Heo et al, 2006). Several cancerspecific mutations in the C2 domain of p110a result in a change to a positive charge, that is, N345K, C378R, E418K, C420R and E453K (Campbell et al, 2004;Samuels et al, 2004;Saal et al, 2005).…”

The RGK family of GTP-binding proteins: Regulators of voltage-dependent calcium channels and cytoskeleton remodeling

“…1 and [56]), however, despite the potential to serve as a site of lipidation, there is no evidence that the cysteine residue is modified, and deletion of the C-7 domain does not alter RGK function [8,48,53,55]. The question of how RGK GTPases localize to the plasma membrane was, at least partially, answered by the recent work of Heo and colleagues illustrating the importance of phosphatidylinositol phosphate (PIP) lipids in the membrane recruitment of RGKs, as well as other Ras-related GTPases containing polybasic tracts in their C-termini [57] (Figs. 1 and 3).…”

“…Instead, plasma membrane localization is critical to RGK-mediated Ca 2+ channel regulation, since anchoring C-terminal Rem [53] or Rem2 truncation mutants [49] to the plasma membrane is sufficient to restore Ca 2+ channel inhibition. As discussed above (see Section 4), the RGK C-terminus would appear to provide an excellent regulatory domain, being subject to both complex patterns of phosphorylation [12,[41][42][43][44][45], serving as the docking site for various binding factors [15,[41][42][43][44][45]80] (Table 1), and as a phosphatidylinositol lipid (PIP) binding domain [53,57] (Fig. 1).…”

Cell surface dynamics – how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton

“…This results in the assembly of domains rich in phosphatidylinositol 3,4,4,5)P 3 ] that act as specialized platforms for plasma membrane dynamics. Protein recruitment to these platforms is not limited to small GTPases and their GEFs, but also includes NPFs, other actin-binding proteins, proteins with polybasic clusters and amphiphylic proteins that are necessary for the deformation of membranes (Heo et al, 2006;Takabayashi et al, 2010; reviewed by Saarikangas et al, 2010;Suetsugu and Gautreau, 2012). The local cooperation between actin remodeling and membrane deformation underlies dynamic processes at the cell surface, causing either protrusion (e.g.…”