Activation of PAR-1 kinase and stimulation of tau phosphorylation by diverse signals require the tumor suppressor protein LKB1 - PubMed (original) (raw)

Comparative Study

Activation of PAR-1 kinase and stimulation of tau phosphorylation by diverse signals require the tumor suppressor protein LKB1

Ji-Wu Wang et al. J Neurosci. 2007.

Abstract

Aberrant phosphorylation of tau is associated with a number of neurodegenerative diseases, including Alzheimer's disease (AD). The molecular mechanisms by which tau phosphorylation is regulated under normal and disease conditions are not well understood. Microtubule affinity regulating kinase (MARK) and PAR-1 have been identified as physiological tau kinases, and aberrant phosphorylation of MARK/PAR-1 target sites in tau has been observed in AD patients and animal models. Here we show that phosphorylation of PAR-1 by the tumor suppressor protein LKB1 is required for PAR-1 activation, which in turn promotes tau phosphorylation in Drosophila. Diverse stress stimuli, such as high osmolarity and overexpression of the human beta-amyloid precursor protein, can promote PAR-1 activation and tau phosphorylation in an LKB1-dependent manner. These results reveal a new function for the tumor suppressor protein LKB1 in a signaling cascade through which the phosphorylation and function of tau is regulated by diverse signals under physiological and pathological conditions.

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Figures

Figure 1.

In vivo phosphorylation of PAR-1 at T408 and its importance in regulating PAR-1 activity. A–D, Scanning electron microscopic images of transgenic fly eyes overexpressing wild-type (WT) PAR-1 (A), PAR-1(T408A) (B), PAR-1(T408E) (C), or wild-type control (D) fly eyes. E, F, Western blot analysis showing that whereas PAR-1 overexpression caused an approximate fourfold increase in wild-type h-tau (E) or h-tau(R406W) (F) phosphorylation at 12E8 sites, PAR-1(T408A) or PAR-1(T408E) had little effects. Tubulin served as a loading control. G, Detection of T408 phosphorylation of wild-type PAR-1 but not PAR-1(T408A) or PAR-1(T408E) produced from transgenes (bottom). Exogenous PAR-1 proteins expressed from the transgenes were tagged with a myc tag and detected with an anti-myc antibody. H, Detection of p-T408-positive endogenous PAR-1 protein from adult fly head extracts prepared from wild-type and hypomorphic PAR-1 (PAR-1W3/PAR-119A) mutant animals. ←, PAR-1-specific bands; *, nonspecific bands.

Figure 2.

LKB1 acts upstream of PAR-1. A–E, Scanning electron microscopic images showing modification of PAR-1-induced eye degeneration by LKB1 overexpression or RNAi knockdown. The eyes of the following genotypes are shown: GMR-Gal4>UAS-PAR-1 (A), GMR-Gal4>UAS-LKB1 (B), GMR-Gal4>UAS-PAR-1/UAS-LKB1 (C), GMR-Gal4>UAS-LKB1 RNAi (D), GMR-Gal4>UAS-PAR-1/UAS-LKB1 RNAi (E). F, Western blot analysis showing increased PAR-1 T408 phosphorylation after LKB1 overexpression. Although LKB1 coexpression also caused an increase in PAR-1 protein, after normalization of total PAR-1 level, there is still a 2.5-fold increase in p-T408 signal. G, Western blot analysis showing decreased PAR-1 T408 phosphorylation after LKB1 knockdown by RNAi. H, Control experiment showing that coexpression of a W RNAi construct had no effect on PAR-1 T408 phosphorylation. I, Western blot analysis demonstrating RNAi knockdown of LKB1 protein expression. W RNAi serves as an RNAi control, and tubulin serves as a loading control.

Figure 3.

Direct phosphorylation of PAR-1 T408 site by LKB1. A, In vitro kinase assay showing phosphorylation of wild-type (WT) PAR-1 but not PAR-1(T408A) recombinant proteins by mammalian LKB1/STRAD/MO25 complex. Top, PAR-1 T408 phosphorylation detected with the p-T408 antibody. Bottom, Coomassie blue staining of the recombinant substrate proteins to show equal loading. B, In vitro kinase assays using wild-type LKB1 and kinase-dead LKB1(LKB1KD), which contains a K194A mutation, to demonstrate the specificity of LKB1 phosphorylation of PAR-1. HEK293 cells were transfected with the indicated FLAG-LKB1 and His-STRAD constructs. Cell lysates were immunoprecipitated with anti-Flag agarose beads, and the immunocomplex was used in in vitro kinase assays using the indicated His-PAR-1 recombinant protein as the substrate. Phosphorylation of PAR-1 was detected with anti-p-T408 antibody. The amounts of immunoprecipitated LKB1, STRAD, and the input PAR-1 substrates were detected by Western blotting with the corresponding anti-epitope tag antibodies.

Figure 4.

Enhancement of h-tau phosphorylation and toxicity by LKB1. A–E, Toluidine blue staining of photoreceptor neurons in transgenic flies overexpressing LKB1 (A) or h-tau(R406W) (B), coexpressing LKB1 and h-tau(R406W) (C), overexpressing h-tau(R406W)S2A (D), or coexpressing LKB1 and h-tau(R406W)S2A (E). Arrows mark ommatidial clusters showing photoreceptor loss. Note that all of the photoreceptor neurons are preserved in A, D, and E; a small number of photoreceptors are lost in B; whereas the majority of the photoreceptor neurons are lost in C. F, Western blot analysis showing increased phosphorylation of h-tau(R406W) at 12E8 and AT8 sites, whereas the phosphorylation at AT180 site was minimally changed after LKB1 coexpression. The T14 antibody recognizes total h-tau. G, Western blot analysis showing no change in tau phosphorylation at the 12E8 site after AMPK coexpression.

Figure 5.

LKB1 mediates the effect of APP and osmotic stress on PAR-1 and tau phosphorylation and toxicity. A–D, Scanning electron microscopic eye images of transgenic flies overexpressing APP695 (A) or PAR-1 (B), coexpressing APP695 and PAR-1(C), or coexpressing APP695, PAR-1, and LKB1 RNAi transgenes (D). E–H, Scanning electron microscopic eye images of transgenic flies overexpressing APP695 (E) or h-tau(R406W) (F), coexpressing APP695 and h-tau(R406W) (G), or coexpressing APP695, h-tau(R406W), and LKB1 RNAi transgenes (H). I, Western blot analysis showing enhancement of PAR-1 T408 phosphorylation by overexpression of wild-type APP or APP containing the London mutation. Wild-type APP and APP(London) caused an approximate twofold and threefold increase in PAR-1 p-T408 levels, respectively. J, Western blot analysis showing attenuation of the effects of APP overexpression on PAR-1 T408 phosphorylation and tau 12E8 site phosphorylation by LKB1 RNAi. Whereas APP caused an approximate threefold increase in PAR-1 p-T408 levels and a twofold increase in h-tau 12E8 signals, LKB1 RNAi attenuated this enhancing effect. K, L, Western blot analyses showing enhancement of PAR-1 T408 phosphorylation (K) or tau 12E8 phosphorylation (L) by NaCl treatment. NaCl treatment caused an approximate fourfold increase in PAR-1 p-T408 level and a twofold increase in h-tau 12E8 phosphorylation. These effects were blocked by LKB1 RNAi but not W RNAi. Tubulin served as a loading control in all panels.

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Activation of PAR-1 kinase and stimulation of tau phosphorylation by diverse signals require the tumor suppressor protein LKB1 - PubMed (original) (raw)