Arabidopsis Phosphatidylinositol Phosphate Kinase 1 Binds F-actin and Recruits Phosphatidylinositol 4-Kinase β1 to the Actin Cytoskeleton

Abstract

The actin cytoskeleton can be influenced by phospholipids and lipid-modifying enzymes. In animals the phosphatidylinositol phosphate kinases (PIPKs) are associated with the cytoskeleton through a scaffold of proteins; however, in plants such an interaction was not clear. Our approach was to determine which of the plant PIPKs interact with actin and determine whether the PIPK-actin interaction is direct. Our results indicate that AtPIPK1 interacts directly with actin and that the binding is mediated through a predicted linker region in the lipid kinase. AtPIPK1 also recruits AtPI4K␤1 to the cytoskeleton. Recruitment of AtPI4K␤1 to F-actin was dependent on the C-terminal catalytic domain of phosphatidylinositol-4-phosphate 5-kinase but did not require the presence of the N-terminal 251 amino acids, which includes 7 putative membrane occupation and recognition nexus motifs. In vivo studies confirm the interaction of plant lipid kinases with the cytoskeleton and suggest a role for actin in targeting PIPKs to the membrane. Phosphatidylinositol phosphate kinases (PIPKs)2 are a family of enzymes that phosphorylate phosphatidylinositol phosphates (PtdInsP) to phosphatidylinositol bisphosphates (PtdInsP 2 ). Arabidopsis has 11 predicted isoforms of PtdInsP kinases (1). AtPIPK1 and AtPIPK10 have been characterized biochemically, and PtdIns4P is the primary substrate making the predominant product phosphatidylinositol (4, 5) bisphosphate (PtdIns(4,5)P 2 ), suggesting that they are similar to the type I PIPKs found in humans (2-5). However, unlike the mammalian, yeast or Caenorhabditis elegans PIPKs, AtPIPK1-9 contain N-terminal putative membrane occupation and recognition nexus (MORN) motifs similar to those first reported in junctophilins (1, 6). MORN motifs are unique to this family of enzymes and have not been reported in any other eukaryotic lipid kinases.In eukaryotic models PtdInsP kinases supply PtdIns(4,5)P 2 for many cellular functions, and it is well known that phospholipids play an integral role in regulating the structure and dynamics of the cytoskeleton through the many actin-binding proteins that interact with PtdIns(4,5)P 2 (7-10). For example, the absence of PtdInsP kinases in yeast has an adverse affect on the yeast cell morphology (11) and causes them to be unable to properly form actin cables (12). In some animal cell lines overexpression of PtdInsP kinases results in the formation of actin comet tails and stress fibers (13, 14) and disruption of membrane trafficking (14). In these animal cell lines, a functional PtdInsP kinase producing PtdIns(4,5)P 2 was necessary to cause the changes in cytoskeletal structure (13,15). In mammalian systems, PIPK activity has been recovered with an F-actin fraction, and the ␣, ␤, and ␥ isoforms have been identified as copurifying with F-actin. The data support a model where these PIPKs do not directly interact with F-actin but that this interaction is mediated by the presence of Racs, small GTP-binding proteins (16). In plants, PtdIns kinase activity (17, 18) and PtdIns...

Increasing Plasma Membrane Phosphatidylinositol(4,5)Bisphosphate Biosynthesis Increases Phosphoinositide Metabolism inNicotiana tabacum

“…We did not detect measurable differences in F-actin morphology based on phalloidin staining in the tobacco cells (see Supplemental Figure 5 online). This may be because the actin binding proteins required to scaffold Hs PIPKIa to actin were not present in the plant cells (Ridley, 2006;Davis et al, 2007). The intensity of phalloidin staining appeared to be consistently greater in the Hs PIPKIa lines, suggesting increased membrane permeability to phalloidin or possibly an increase in cable thickness reminiscent of the Arabidopsis fra3 mutant.…”

A Comprehensive Survey of Retracted Articles from the Scholarly Literature

“…However, many records in both databases fail to indicate the retracted status of articles. For example, some, e.g., [22] are marked as retracted in WoS but not in PubMed (when checked on 7 Jun 2012). Thus, determining the true proportions of retracted articles in these databases would require surveying records for all articles known to be retracted.…”

“…To compile a comprehensive list of retracted articles, a wide variety of data sources must be consulted for several reasons. Bibliographic databases vary with respect to the journals they index “cover-to-cover,” the journals they index only selectively, and their policies regarding retroactively marking existing records for retracted publications [21], [22]. Due to the latter two factors, even if database X covers journal Y, identifying the retracted articles from that journal in the database may not be possible.…”

At the poles across kingdoms: phosphoinositides and polar tip growth

“…PI4P 5-kinases have been found to be associated with the plasma membrane of animal, yeast, and also of A. thaliana and tobacco cells using fluorescent reporters (Lee et al 2007; Ischebeck et al 2008; Kusano et al 2008; Sousa et al 2008; Stenzel et al 2008). Enzyme activities have, however, been detected not only in enriched plasma membrane preparations (Sommarin and Sandelius 1988; Perera et al 1999; Heilmann et al 2001; Kobayashi et al 2005; Santarius et al 2006), but also in other subcellular fractions such as the nucleus of mammalian and yeast cells (Ciruela et al 2000; Audhya and Emr 2003; Santarius et al 2006), the actin cytoskeleton of yeast and plants (Desrivieres et al 1998; Doughman et al 2003; Davis et al 2007), and endomembranes of animal and plant cells (Whatmore et al 1996; Heilmann et al 1999; Im et al 2007a). The mechanism recruiting PI4P 5-kinases to the plasma membrane in plants is unknown; however sphingolipids have been shown to be involved in plasma membrane localization of Mss4p, the only PI4P 5-kinase present in yeast (Kobayashi et al 2005).…”