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MARK/PAR1 kinase is a regulator of microtubule-dependent transport in axons.

Mandelkow EM, Thies E, Trinczek B, Biernat J, Mandelkow E - J. Cell Biol. (2004)

Bottom Line: The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks.This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged.This transport inhibition can be rescued by phosphorylating tau with MARK.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck Unit for Structural Molecular Biology, 22607 Hamburg, Germany. mandelkow@mpasmb.desy.de

ABSTRACT
Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.

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Recovery of anterograde movements of mitochondria by coexpression of tau and MARK2. (a,1–3) Axons of RGCs stained for mitochondria (MitoTracker, a 1), CFP-tau (a 2), YFP-MARK2 (a 3). Numerous mitochondria are present in axons, in spite of the presence of tau, because the phosphorylation of tau by MARK detaches it from microtubules and facilitates transport (closed arrowheads). (b) Velocity histogram of mitochondria. Note the substantial recovery of anterograde transport (dark blue bars) after coexpression with MARK2 (compare with Fig. 5 k, transfection of tau only). Error bars indicate SEM. (c, 1–4) Axons showing YFP-MARK2 (c 1) and phospho-Tau (at KXGS motifs, antibody 12E8, c 2, closed arrowheads). Axons after transfection with inactive CFP-MARK2 (c 3), which fails to increase the phosphorylation of KXGS motifs of tau (c 4, antibody 12E8 shows only background staining). (d, 1–3) Axons after transfection of cells with YFP-MARK2 (d 1) and then extracted with Triton X-100 and stained for total Tau (antibody K9JA, d 2) phospho-Tau (antibody 12E8, d 3). Note that Tau phosphorylated at KXGS motifs disappears because it is not attached to microtubules (closed arrowheads). (e, 1 and 2) Axons not transfected with MARK, extracted with Triton X-100, show strong staining with Tau (K9JA, e 1) but not with phospho-Tau (12E8, e 2).
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fig6: Recovery of anterograde movements of mitochondria by coexpression of tau and MARK2. (a,1–3) Axons of RGCs stained for mitochondria (MitoTracker, a 1), CFP-tau (a 2), YFP-MARK2 (a 3). Numerous mitochondria are present in axons, in spite of the presence of tau, because the phosphorylation of tau by MARK detaches it from microtubules and facilitates transport (closed arrowheads). (b) Velocity histogram of mitochondria. Note the substantial recovery of anterograde transport (dark blue bars) after coexpression with MARK2 (compare with Fig. 5 k, transfection of tau only). Error bars indicate SEM. (c, 1–4) Axons showing YFP-MARK2 (c 1) and phospho-Tau (at KXGS motifs, antibody 12E8, c 2, closed arrowheads). Axons after transfection with inactive CFP-MARK2 (c 3), which fails to increase the phosphorylation of KXGS motifs of tau (c 4, antibody 12E8 shows only background staining). (d, 1–3) Axons after transfection of cells with YFP-MARK2 (d 1) and then extracted with Triton X-100 and stained for total Tau (antibody K9JA, d 2) phospho-Tau (antibody 12E8, d 3). Note that Tau phosphorylated at KXGS motifs disappears because it is not attached to microtubules (closed arrowheads). (e, 1 and 2) Axons not transfected with MARK, extracted with Triton X-100, show strong staining with Tau (K9JA, e 1) but not with phospho-Tau (12E8, e 2).

Mentions: The next question was whether the inhibitory effect of tau could be relieved by removing tau from microtubules. We transfected RGCs with CFP-tau and YFP-MARK2, both proteins were expressed and became distributed along the axons (Fig. 6 a, 1–3). This has striking consequences for mitochondrial movements (Fig. 6 b): anterograde movements became substantially reactivated (∼30%); the fraction of stationary particles decreased (compare Fig. 6 b with Fig. 5 k); and the density of particles increased again. A strong reaction of the 12E8 antibody appeared, revealing that tau was phosphorylated at the KXGS motifs (Fig. 6 c, 2). The phosphorylation of tau was accompanied by its removal from microtubules: if cells transfected with tau and MARK2 (Fig. 6 d, 1) were extracted with Triton X-100 the reaction with antibody 12E8 was lost (Fig. 6 d, 3), indicating that the phosphorylated tau was indeed detached from microtubules, and only traces of unphosphorylated tau remained in the axon (Fig. 6 d, 2). This is in strong contrast to cells not transfected with MARK where extraction by Triton X-100 does not remove tau from microtubules (Fig. 6 e, 1) and tau remains unphosphorylated at KXGS motifs (Fig. 6 e, 2). We conclude that the blockade of traffic was relieved after removing tau from the tracks by phosphorylation at the KXGS motifs. Thus, MARK counteracts the inhibitory effect of tau. This interpretation was checked by several controls. Transfection of RGCs with GFP had only a minor effect, most mitochondria moved anterogradely (∼50%), only a small fraction were in the pause state (∼30%), underscoring that GFP is a neutral marker (unpublished data). The same was true for the kinase-dead mutant of MARK2 (Fig. 6 c, 3), indicating that the effect of MARK was due to its kinase activity. Like active MARK2 or GFP, the inactive mutant was also distributed evenly along the axon (Fig. 6 c, 3), and there was no phosphorylation of tau in the repeat domain above background (judging by 12E8 immunofluorescence; Fig. 6 c, 4). Finally, we constructed an adenovirus vector encoding a mutant CFP-tau where all four KXGS motifs were changed into KXGA so that the targets of MARK2 were eliminated. Transfection in RGCs caused a strong inhibition of transport, similar to wild-type tau (compare Fig. 7, a and d with Fig. 5, j and k). However, in this case the cotransfection with MARK2 was not able to induce phosphorylation of tau or to rescue the mobility of mitochondria (compare Fig. 7, b, c, and e with Fig. 6 b), showing that blockage of traffic by tau and rescue by MARK2 requires phosphorylatable KXGS motifs on tau.


MARK/PAR1 kinase is a regulator of microtubule-dependent transport in axons.

Mandelkow EM, Thies E, Trinczek B, Biernat J, Mandelkow E - J. Cell Biol. (2004)

Recovery of anterograde movements of mitochondria by coexpression of tau and MARK2. (a,1–3) Axons of RGCs stained for mitochondria (MitoTracker, a 1), CFP-tau (a 2), YFP-MARK2 (a 3). Numerous mitochondria are present in axons, in spite of the presence of tau, because the phosphorylation of tau by MARK detaches it from microtubules and facilitates transport (closed arrowheads). (b) Velocity histogram of mitochondria. Note the substantial recovery of anterograde transport (dark blue bars) after coexpression with MARK2 (compare with Fig. 5 k, transfection of tau only). Error bars indicate SEM. (c, 1–4) Axons showing YFP-MARK2 (c 1) and phospho-Tau (at KXGS motifs, antibody 12E8, c 2, closed arrowheads). Axons after transfection with inactive CFP-MARK2 (c 3), which fails to increase the phosphorylation of KXGS motifs of tau (c 4, antibody 12E8 shows only background staining). (d, 1–3) Axons after transfection of cells with YFP-MARK2 (d 1) and then extracted with Triton X-100 and stained for total Tau (antibody K9JA, d 2) phospho-Tau (antibody 12E8, d 3). Note that Tau phosphorylated at KXGS motifs disappears because it is not attached to microtubules (closed arrowheads). (e, 1 and 2) Axons not transfected with MARK, extracted with Triton X-100, show strong staining with Tau (K9JA, e 1) but not with phospho-Tau (12E8, e 2).
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fig6: Recovery of anterograde movements of mitochondria by coexpression of tau and MARK2. (a,1–3) Axons of RGCs stained for mitochondria (MitoTracker, a 1), CFP-tau (a 2), YFP-MARK2 (a 3). Numerous mitochondria are present in axons, in spite of the presence of tau, because the phosphorylation of tau by MARK detaches it from microtubules and facilitates transport (closed arrowheads). (b) Velocity histogram of mitochondria. Note the substantial recovery of anterograde transport (dark blue bars) after coexpression with MARK2 (compare with Fig. 5 k, transfection of tau only). Error bars indicate SEM. (c, 1–4) Axons showing YFP-MARK2 (c 1) and phospho-Tau (at KXGS motifs, antibody 12E8, c 2, closed arrowheads). Axons after transfection with inactive CFP-MARK2 (c 3), which fails to increase the phosphorylation of KXGS motifs of tau (c 4, antibody 12E8 shows only background staining). (d, 1–3) Axons after transfection of cells with YFP-MARK2 (d 1) and then extracted with Triton X-100 and stained for total Tau (antibody K9JA, d 2) phospho-Tau (antibody 12E8, d 3). Note that Tau phosphorylated at KXGS motifs disappears because it is not attached to microtubules (closed arrowheads). (e, 1 and 2) Axons not transfected with MARK, extracted with Triton X-100, show strong staining with Tau (K9JA, e 1) but not with phospho-Tau (12E8, e 2).
Mentions: The next question was whether the inhibitory effect of tau could be relieved by removing tau from microtubules. We transfected RGCs with CFP-tau and YFP-MARK2, both proteins were expressed and became distributed along the axons (Fig. 6 a, 1–3). This has striking consequences for mitochondrial movements (Fig. 6 b): anterograde movements became substantially reactivated (∼30%); the fraction of stationary particles decreased (compare Fig. 6 b with Fig. 5 k); and the density of particles increased again. A strong reaction of the 12E8 antibody appeared, revealing that tau was phosphorylated at the KXGS motifs (Fig. 6 c, 2). The phosphorylation of tau was accompanied by its removal from microtubules: if cells transfected with tau and MARK2 (Fig. 6 d, 1) were extracted with Triton X-100 the reaction with antibody 12E8 was lost (Fig. 6 d, 3), indicating that the phosphorylated tau was indeed detached from microtubules, and only traces of unphosphorylated tau remained in the axon (Fig. 6 d, 2). This is in strong contrast to cells not transfected with MARK where extraction by Triton X-100 does not remove tau from microtubules (Fig. 6 e, 1) and tau remains unphosphorylated at KXGS motifs (Fig. 6 e, 2). We conclude that the blockade of traffic was relieved after removing tau from the tracks by phosphorylation at the KXGS motifs. Thus, MARK counteracts the inhibitory effect of tau. This interpretation was checked by several controls. Transfection of RGCs with GFP had only a minor effect, most mitochondria moved anterogradely (∼50%), only a small fraction were in the pause state (∼30%), underscoring that GFP is a neutral marker (unpublished data). The same was true for the kinase-dead mutant of MARK2 (Fig. 6 c, 3), indicating that the effect of MARK was due to its kinase activity. Like active MARK2 or GFP, the inactive mutant was also distributed evenly along the axon (Fig. 6 c, 3), and there was no phosphorylation of tau in the repeat domain above background (judging by 12E8 immunofluorescence; Fig. 6 c, 4). Finally, we constructed an adenovirus vector encoding a mutant CFP-tau where all four KXGS motifs were changed into KXGA so that the targets of MARK2 were eliminated. Transfection in RGCs caused a strong inhibition of transport, similar to wild-type tau (compare Fig. 7, a and d with Fig. 5, j and k). However, in this case the cotransfection with MARK2 was not able to induce phosphorylation of tau or to rescue the mobility of mitochondria (compare Fig. 7, b, c, and e with Fig. 6 b), showing that blockage of traffic by tau and rescue by MARK2 requires phosphorylatable KXGS motifs on tau.

Bottom Line: The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks.This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged.This transport inhibition can be rescued by phosphorylating tau with MARK.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck Unit for Structural Molecular Biology, 22607 Hamburg, Germany. mandelkow@mpasmb.desy.de

ABSTRACT
Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.

Show MeSH
Related in: MedlinePlus