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A PIP5 kinase essential for efficient chemotactic signaling.

ABSTRACT: In neutrophils and Dictyostelium, chemoattractant gradients generate directed cell migration by eliciting signaling events that bias intrinsic motility and favor the production and retention of upgradient pseudopods. Phosphoinositides are actively regulated during chemotaxis in these cells, most iconically in the production of PI(3,4,5)P3 gradients within the plasma membrane. Although it is now known that PI(3,4,5)P3 signaling is nonessential for gradient sensing, the role of the related phosphoinositide PI(4,5)P2 is little understood, despite its clear importance in many cell biological processes. We describe here a PIP5 kinase, PikI, which produces PI(4,5)P2 and is essential for efficient chemotaxis of Dictyostelium cells. Without PikI, PI(4,5)P2 levels are reduced by 90%, and while pikI(-) cells move at normal speeds, they are highly disorientated in cAMP gradients. Following chemotactic stimulation, Ras is efficiently activated in pikI(-) cells, yet Ras-dependent responses (including activation of PKB) are severely impaired. PikI is phosphorylated by PKB, and in vitro studies of a phosphomimic mutant suggest that this phosphorylation increases PikI activity. We propose that adequate PI(4,5)P2 levels are required to couple activated Ras to its downstream effectors and that these levels are regulated by PikI, making it a crucial player in gradient sensing.

SUBMITTER: Fets L

PROVIDER: S-EPMC3969243 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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A PIP5 kinase essential for efficient chemotactic signaling.

Fets Louise L Nichols John M E JM Kay Robert R RR

Current biology : CB 20140130 4

In neutrophils and Dictyostelium, chemoattractant gradients generate directed cell migration by eliciting signaling events that bias intrinsic motility and favor the production and retention of upgradient pseudopods. Phosphoinositides are actively regulated during chemotaxis in these cells, most iconically in the production of PI(3,4,5)P3 gradients within the plasma membrane. Although it is now known that PI(3,4,5)P3 signaling is nonessential for gradient sensing, the role of the related phospho ...[more]

PMID: 24485835

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Project description:Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit swapping interactions that will need to be taken into account in experimental applications of single-chain chemoreceptors.

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A PIP5 kinase essential for efficient chemotactic signaling.

Publications

A PIP5 kinase essential for efficient chemotactic signaling.

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