Localization of a Binding Site for Phosphatidylinositol 4,5-Bisphosphate on Human Profilin

Abstract

Profilin is a small 12-15-kDa actin-binding protein, which in eukaryotic organisms is ubiquitous and necessary for normal cell growth and function. Although profilin's interactions with its three known ligands (actin monomers, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), and poly-L-proline (PLP)) have been well characterized in vitro, its precise role in cells remains largely unknown. By binding to clusters of PIP 2 , profilin is able to inhibit the hydrolysis of PIP 2 by phospholipase C␥1 (PLC␥1). This ability is the result of profilin's affinity for PIP 2 , but the specific residues of profilin's amino acid sequence involved in the binding of PIP 2 are not known. Using site-directed mutagenesis, we sought to localize regions of profilin important for this interaction by generating the following mutants of human profilin (named according to the wild-type amino acid altered, its position, and the amino acid substituted in its place): Y6F, D8A, L10R, K25Q, K53I, R74L, R88L, R88L/K90E, H119D, G121D, and K125Q. With the exception of L10R, all of the mutants were successfully expressed in Escherichia coli and purified by affinity chromatography on PLP-Sepharose. Only Y6F and K25Q demonstrated moderately less stringent binding to PLP, indicating that most of the mutations did not induce marked alterations of profilin's structure. When tested for their relative abilities to inhibit the hydrolysis of PIP 2 by PLC␥1, most of the mutants were indistinguishable from wild-type profilin. Exceptions included D8A, which demonstrated increased inhibition of PLC␥1, and R88L, which demonstrated decreased inhibition of PLC␥1. To assess the importance of the region surrounding residue 88 of human profilin, three synthetic decapeptides selected to correspond to non-overlapping stretches of the human profilin sequence were tested for their abilities to inhibit PLC␥1. We found that only the decapeptide that matched the peptide stretch centered around residue 88 was able to inhibit PLC␥1 activity substantially and was able to do so at nearly wild-type profilin levels. Taken together with the finding that mutating residue 88 resulted in decreased inhibition of PLC␥1 activity, these data provide strong evidence that this region of human profilin represents an important binding site for PIP 2 .Since the molecule's discovery 20 years ago (1), profilin's interactions with its three ligands (actin monomers, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), 1 and PLP) have been well established through in vitro studies (for review, see Ref.2). For example, by binding to actin monomers in a 1:1 complex, profilin decreases the critical concentration of monomeric actin in the presence of thymosin ␤4 (3), inhibits the spontaneous nucleation of actin filaments (4), and catalyzes the exchange of adenosine nucleotides bound to actin monomers (5). By binding to PIP 2 and to a lesser degree its precursor PIP (6), profilin prevents PLC␥1 from hydrolyzing PIP 2 (7). However, when PLC␥1 is phosphorylated on specific tyrosine residues, such as it occurs...

Profilin1 regulates PI(3,4)P 2 and lamellipodin accumulation at the leading edge thus influencing motility of MDA-MB-231 cells

“…Table S1 summarizes how each of these mutations affects various ligand interactions of profilin1, as determined by quantitative and nonquantitative binding assays in previous studies [only phosphoinositide binding of H119E-profilin1 was not specifically tested previously; we qualitatively confirmed that H119E substitution does not impair profilin1's phosphoinositide binding (Fig. S1), and this is consistent with a previously reported finding for the analogous H119D mutant (15)]. In summary, H119E and H133S substitutions selectively abolish profilin1's interaction with actin (25-fold lower binding than WT) and polyproline ligands (50-fold lower binding than WT), respectively (16).…”

“…It was previously reported that phosphoinositide binding of profilin1 can inhibit phospholipase-Cγ (PLCγ)-mediated PI(4,5)P 2 hydrolysis in vitro (7,15). These findings led to a speculation that profilin1 could be a phosphoinositide regulator, but it was never confirmed in vivo.…”

“…In summary, H119E and H133S substitutions selectively abolish profilin1's interaction with actin (25-fold lower binding than WT) and polyproline ligands (50-fold lower binding than WT), respectively (16). R88L substitution not only causes a major defect in phosphoinositide binding of profilin1 [its affinity for PI(4,5)P 2 is threefold lower than WT] (15,17), but also severely impairs profilin1's ability to inhibit actin polymerization and exchange nucleotide on actin in vitro (15). This implies that R88L-profilin1 is also defective in actin binding (this is true for all other phosphoinositide mutants of profilin1 identified to date and results from likely overlap between actin and phosphoinositide binding sites within profilin1).…”

Pollen Profilin Function Depends on Interaction with Proline-Rich Motifs

“…Eight of the most highly conserved residues in eukaryotic profilins ( Thorn et al, 1997), marked with asterisks in Figure 1, have been implicated in PLP binding by using mutagenesis (Björkegren et al, 1993;Haarer et al, 1993), nuclear magnetic resonance spectroscopy (Metzler et al, 1994; Domke et al, 1997), and x-ray crystallography (Mahoney et al, 1997). The hydroxyl group of tyrosine-6 was shown to interact directly with the backbone of the PLP peptide (Mahoney et al, 1997), and the Y6F mutation in human profilin I was shown to elute from PLP-agarose at a lower concentration of urea than did the wild-type protein (Sohn et al, 1995). Therefore, the observation that the ZmPRO1-Y6F mutant has higher affinity for PLP is somewhat surprising.…”

Profilin, and Vascular Diseases

“…The binding domains of actin and actin related proteins (blue; Schutt et al, 1993) and PtdIns 4,5-P2 (red; Sohn et al, 1995) overlap, while that for proline-cluster sequences (green; Metzler et al, 1994) is located at the opposite side of the profilin molecule, adapted from Schlüter et al, (1997) with permission.…”