Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jul;69(7):514-27.
doi: 10.1002/cm.21021. Epub 2012 Mar 7.

Dyrk kinases regulate phosphorylation of doublecortin, cytoskeletal organization, and neuronal morphology

Tatiana I Slepak  1 , Lindsey D Salay, Vance P Lemmon, John L Bixby
Affiliations

Dyrk kinases regulate phosphorylation of doublecortin, cytoskeletal organization, and neuronal morphology

Tatiana I Slepak et al. Cytoskeleton (Hoboken). 2012 Jul.

Abstract

In a neuronal overexpression screen focused on kinases and phosphatases, one "hit" was the dual specificity tyrosine phosphorylation-regulated kinase (Dyrk4), which increased the number of dendritic branches in hippocampal neurons. Overexpression of various Dyrk family members in primary neurons significantly changed neuronal morphology. Dyrk1A decreased axon growth, Dyrk3 and Dyrk4 increased dendritic branching, and Dyrk2 decreased both axon and dendrite growth and branching. Kinase-deficient mutants revealed that most of these effects depend on kinase activity. Because doublecortin (DCX), a microtubule-binding protein, regulates cytoskeletal dynamics and neuronal morphogenesis, we investigated the possibility that DCX is a target of Dyrks. We found that overexpression of Dyrk2 and Dyrk3, but not Dyrk1A or Dyrk4, can change DCX phosphorylation status. Mutation of a consensus phosphorylation site for Dyrk kinases at Serine 306 (Ser306) in DCX indicated that this is one target site for Dyrk2 and Dyrk3. Overexpression of Dyrk2 restored altered DCX distribution in the growth cones of dendrites and axons, and partially reversed the morphological effects of DCX overexpression; some of these effects were abrogated by mutation of Ser306 to alanine. These studies implicate Dyrks in the regulation of cytoskeletal organization and process outgrowth in neurons, and suggest that DCX is one relevant Dyrk target.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. Subcellular localization of Dyrks in transfected hippocampal neurons
(A–D) E18 hippocampal neurons were transfected with 2A-tagged Dyrks, cultured for 5 days, and stained with anti-beta III tubulin (green) and anti-2A (red) antibodies. Arrows point to transfected cells. Scale bar is 50 μm (5μm in the inset). (E–H) 2A (white) and nuclear (DAPI, blue) staining only of the merged images. The insets show a magnified image of the cell body of the transfected cell. Dyrk1A (E) localizes in speckled structures of the nucleus. Dyrk2 (F), Dyrk3 (G), and Dyrk4 (H) are cytosolic, present in dendrites (Dyrk3 and 4), or both dendrites and axons (Dyrk2), and are not detected in the nucleus.
Fig. 2
Fig. 2. Effect of DYRKs on neuron morphology
Primary neurons were transfected with Dyrk1A (A), Dyrk2 (B), Dyrk3 (C), or Dyrk4 (D) plasmids, either WT or kinase-deficient (KD), or with control mCherry plasmids. Transfected neurons (N ≥ 30 per condition) were manually traced (using the tubulin channel) to measure axonal (Ax) total length and branching, and dendritic (Dn) total length and branching. All measured parameters were normalized to the control. Dyrk1A overexpression significantly decreased axonal length (A). Dyrk2 overexpression decreased all 4 measured parameters, and the KD mutant increased dendritic length and branching (B). Dyrk3 overexpression increased dendritic branching, and the KD mutant tended to decrease this parameter. KD Dyrk3 also increased axonal length (C). Dyrk4 overexpression, but not the KD mutant, increased dendritic branching (D). Graphs show Mean ± SEM. *, p < 0.05; **, p< 0.01
Fig. 3
Fig. 3. Dyrk1A and Dyrk2 overexpression affect subcellular localization of DCX
(A–D) In non-transfected neurons DCX is in the cell body and is enriched at the neurite tips between actin filaments (B) and microtubules (C and E, J, arrowheads). (F–I) Merged images of neurons transfected with 2A-tagged mCherry control or Dyrks and stained with anti-2A (magenta), anti-beta III tubulin (green) and anti-DCX (red) antibodies. (K–N) The same images with DCX staining only. Overexpression of Dyrk1A (F, K) or Dyrk2 (G, L) alter DCX localization compared to control (E, J). Overexpression of Dyrk3 (H, M) or Dyrk4 (I, N) does not obviously affect DCX localization. Arrows show DCX staining in transfected neurons; arrows, in untransfected cells. Scale bar is 20 μm.
Fig. 4
Fig. 4. Dyrk2 and Dyrk3 affect DCX phosphorylation
(A) Amino acid alignment of a C-terminal fragment of DCX from human (hDCX), rat, and mouse (mDCX). Highlighted in yellow is a putative consensus sequence for Dyrk phosphorylation containing Ser306 (left arrow). Phosphorylation sites in DCX targeted by known S-T kinases are shown in blue. Right arrow shows position of Ser327, a target site for GSK3beta (Bilimoria et al., 2010) (B, C) HEK293 cells were co-transfected with DCX(WT), DCX(S306A), or DCX(S327A) and either control (GFP) plasmid or plasmids expressing Dyrk1A (D1A), Dyrk2 (D2), Dyrk3 (D3), or Dyrk4 (D4). Western blots of cell lysates were performed with anti-pan DCX (DCX ab, B), or with a phospho-specific antibody raised against DCX(Ser334) (C, bolded and underlined in A). Arrows in B point to various phosphorylated bands of DCX. M, molecular weight markers.
Fig. 5
Fig. 5. Dyrk2 reverses the DCX overexpression phenotype
Hippocampal neurons were co-transfected with 2A-tagged DCX together with control plasmid (mCherry, Aa) or Dyrk1A (Ab), Dyrk2 (Ac), Dyrk3 (Ad), or Dyrk4 (Ae) and cultured for 5 days. (A) Transfected cells identified by staining with anti-2A antibody (red) and marked by arrows. Dyrk2, but not other Dyrks, reverted the DCX overexpression phenotype (Ac). Scale bar is 100 μm. (B) Graphs of axon total length (Ba), dendrite total length (Bb), axon branching (Bc), and dendrite branching (Bd). Means ± SEM; N ≥30 cells. White bars, cells transfected with Dyrks only; black bars, cells transfected with Dyrks + DCX. *, p <0.05; **, p< 0.01; ***; p< 0.001; ns, not significant.
Fig. 6
Fig. 6. Effect of Dyrks on the DCX-induced “curviness” phenotype
Axon and dendrite curviness were assayed by measuring tortuosity (see text), the lowest possible value of which is 1 (straight line). Hippocampal neurons were transfected with plasmids expressing various Dyrks together with mCherry (mCh, control) or Dyrk1A (D1A), Dyrk2 (D2), Dyrk3 (D3), or Dyrk4 (D4). A and B show the average tortuosity (Mean ± SEM) for axons and dendrites, respectively. DCX overexpression significantly increased tortuosity of axons but not dendrites. Co-expression of Dyrk1A, Dyrk3, or Dyrk4 with DCX increases DCX-induced tortuosity in both axons and dendrites, whereas co-expression of Dyrk2 reduces the tortuosity to that of control. All neurites were measured for N≥30 cells. *, p <0.05; **, p< 0.01; ***; p< 0.001; ns, not significant.
Fig. 7
Fig. 7. Ser306 of DCX is not required for the Dyrk2 effect on the DCX phenotype
Neurons were transfected with 2A-tagged Dyrk2 (A, F, K), 2A-tagged wild type DCX (DCX) (B, G, L), or the DCX(S306A) mutant (C, H, M), or co-transfected with Dyrk2 + DCX (D, I, N) or Dyrk2 + DCX(S306A) (E, J, O). Cells were cultured for 5 days and stained with anti-β3 tubulin (Tubulin, A–E), and anti-doublecortin (DCX, F) or anti-2A (2A, G–J). Arrowheads point to localization of DCX and tubulin. Overexpression of Dyrk2 displaces endogenous DCX from neurites (A, F, K). Overexpressed WT DCX (B, G, L) or DCX(S306A) mutant (C, H, M) show co-localized staining with tubulin along the length of a neurite. The DCX(S306A) mutant produces even more curved neurites (C, H, M) compared to WT DCX (curved neurites traced by asterisks in G). Co-transfection of Dyrk2 with either WT or mutant DCX shortens dendrites and partially restores normal distribution of DCX in neurites where it is enriched at the tips (D, I, N and E, J, O). Inset in panel O shows transfected neuron (arrow) at the lower magnification to show similarity in overall morphology between cells co-transfected with DCX(S306A) and Dyrk2 (red) and untransfected cells (green). Scale bar, 20 μm; 100 μm, inset.
Fig. 8
Fig. 8. Ser306 of DCX is required for Dyrk2 reversion of the axonal, but not the dendritic DCX overexpression phenotype
Hippocampal neurons were co-transfected with wild type 2A-tagged DCX (WT) or DCX S306A together with control plasmid (mCherry) or Dyrk2 (D2). After 5 days, neurons were fixed, stained, and analyzed for axon length (A), dendrite length (B), axon branching (C), and dendrite branching (D). Dyrk2 decreased dendrite length, and increased axon length and branching, in wt DCX-expressing neurons (see also Fig. 5). Dyrk2 also decreased dendrite length in DCX S306A-expressing neurons, but did not affect axon length or axon branching. Means ± SEM; N≥30 cells. *, p <0.05; **, p< 0.01; ***; p< 0.001; ns, not significant.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alvarez M, Altafaj X, Aranda S, de la Luna S. DYRK1A autophosphorylation on serine residue 520 modulates its kinase activity via 14-3-3 binding. Molecular biology of the cell. 2007;18:1167–1178. - PMC - PubMed
    1. Alvarez M, Estivill X, de la Luna S. DYRK1A accumulates in splicing speckles through a novel targeting signal and induces speckle disassembly. Journal of cell science. 2003;116:3099–3107. - PubMed
    1. Aranda S, Laguna A, de la Luna S. DYRK family of protein kinases: evolutionary relationships, biochemical properties, and functional roles. The FASEB journal: official publication of the Federation of American Societies for Experimental Biology 2010 - PubMed
    1. Becker W, Joost HG. Structural and functional characteristics of Dyrk, a novel subfamily of protein kinases with dual specificity. [Accessed October 29, 2011];Progress in nucleic acid research and molecular biology. 1999 62:1–17. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9932450. - PubMed
    1. Bielas SL, Serneo FF, Chechlacz M, Deerinck TJ, Perkins GA, Allen PB, Ellisman MH, Gleeson JG. Spinophilin facilitates dephosphorylation of doublecortin by PP1 to mediate microtubule bundling at the axonal wrist. Cell. 2007;129:579–591. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources