Stress‐induced tyrosine phosphorylation of actin in Dictyostelium cells and localization of the phosphorylation site to tyrosine‐53 adjacent to the DNase I binding loop

Abstract

Actin is known to be phosphorylated at tyrosine, serine, or threonine residues in various cells. In cells of Dictyosteliura discoideum, a rise in the tyrosine phosphorylation of actin is observed in response to ATP depletion. An actin fraction rich in phosphotyrosine was obtained by chromatography on the weak anion exchanger Mono-P. Mass spectrometry and amino acid sequencing of protease cleavage products indicated that a single tyrosine residue was phosphorylated. Localization of this residue to position 53 of the actin sequence attributed the modification to a site that is critical for the capability of actin to polymerize. Induction of the tyrosine phosphorylation by heat shock and Cd 2+ ions indicates that this modification of actin is implicated in the response of Dictyostelium cells to stress.

Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding

“…In none of these examples, however, is the precise connection between actin phosphorylation and cytoskeleton remodeling well understood. Such a connection is better established in Dictyostelium cells, in which the developmental cycle correlates closely with the extent of actin tyrosine phosphorylation (6)(7)(8)(9)(10)(11).…”

“…At this high level of actin phosphorylation, the spores of the mature fruiting bodies remain viable for Ϸ20 days, at which time viability and actin phosphorylation levels both decrease, disappearing entirely by Ϸ30 days. Increases in actin phosphorylation also occur when vegetative cells are exposed to heat stress, sodium azide, and the phosphotyrosine phosphatase inhibitor phenylarsine oxide (6,8,11,13).…”

“…The phosphorylation site on Dictyostelium actin has been mapped to residue Tyr-53 (8,11). This site is near the DNase I-binding loop (D-loop) of actin (residues 40-50), which is implicated in intersubunit contacts in the filament (14)(15)(16)(17).…”

Phosphorylation of actin Tyr-53 inhibits filament nucleation and elongation and destabilizes filaments

“…Tyrosine phosphorylation also occurs when vegetative amoebae in nutrient medium are exposed to phenylarsine oxide (PAO) (5), an inhibitor of phosphotyrosine phosphatase, or are subjected to stress, for example, inhibition of oxidative phosphorylation (8,9) or elevated temperature (9), with parallel changes in cell shape similar to the changes that occur when cells are transferred from nonnutrient to nutrient medium.…”

DictyosteliumStress-activated Protein Kinase α, a Novel Stress-activated Mitogen-activated Protein Kinase Kinase Kinase-like Kinase, Is Important for the Proper Regulation of the Cytoskeleton

“…This remodeling includes the rearrangement of F-actin and myosin II fibers at the cell cortex to form the core of a rigid network resembling a shell-like structure, thereby providing the physical strength to resist the osmotic shock (Insall, 1996). Other known cytoskeletal changes activated by osmotic shock in Dictyostelium include tyrosine phosphorylation of actin, threonine phosphorylation of myosin II, and an increase in the levels of RMLC (regulatory myosin light chain) and cofilin and a decrease in DdLIM in the cortex (Howard et al, 1993;Jungbluth et al, in 1994Jungbluth et al, in , 1995Aizawa et al, 1999;Gamper et al, 1999;Zischka et al, 1999). In addition, the F-actin cross-linking protein ␣-actinin and the 120-kDa gelation factor are important for protection of Dictyostelium cells from osmotic stress and support the pivotal role the cytoskeleton plays in the osmotic response (Rivero et al, 1996).…”