Unrooted neighbor joining tree of human GCKs MST4, STK24, STK25, MINK1 and their orthologues in ascomycetes.

<p>Catalytic domains of the proteins were aligned with ClustalX2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139163#pone.0139163.ref065" target="_blank">65</a>]. The human GCKs are displayed in yellow, the orthologues from the yeast <i>S</i>. <i>pombe</i> and <i>S</i>. <i>cerevisiae</i> in orange. SmKIN3-like kinases and SmKIN-24-like kinases from filamentous ascomycetes are shown in green. Accession numbers of the proteins are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139163#pone.0139163.g002" target="_blank">Fig 2</a> except of the <i>Aspergillus nidulans</i> GCKs AnSEPL (C8V5Z7) and An5674 (Q5B1A6). The number at the nodes indicates bootstrap support of 1000 iterations.</p

BLASTP search of the human STRIPAK associated GCKs against the <i>S</i>. <i>macrospora</i> proteom.

<p>BLASTP search of the human STRIPAK associated GCKs against the <i>S</i>. <i>macrospora</i> proteom.</p

Strains used for this study.

<p>ect = ectopically integrated, FL = full length, nat = nourseothricin-resistance cassette, hph = hygromycin-resistance cassette, ssi = single- spore isolate, hyg^R = hygromycin resistant, nat^R = nourseothricin resistant</p><p>Strains used for this study.</p

Multiple sequence alignment and amino-acid identity of mammalian kinases identified as STRIPAK members with putative homologs from Ascomycota.

<p>Protein sequences were aligned with ClustalX2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139163#pone.0139163.ref065" target="_blank">65</a>] and visualized with GeneDoc [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139163#pone.0139163.ref070" target="_blank">70</a>]. MST4, mammalian STE20-like protein kinase 4 (Accession number: Q9P289); STK24, mammalian STE20-like protein kinase 3 (Q9Y6E0); STK25, mammalian serine/threonine-protein kinase 25 (O00506); MINK1, mammalian misshapen-like kinase 1 (Q8N4C8); SmKIN24, putative serine/threonine-protein kinase 24 from <i>Sordaria macrospora</i> (F7VQV9), NcSTK-24 (STK-6, MST-1), serine/threonine-protein kinase 24 from <i>Neurospora crassa</i> (V5IQF9); Fg ESU11740, serine/threonine-protein kinase 24 from <i>Fusarium graminearum</i> (I1RNY7); Pc21g04360, putative STE20-like protein kinase from <i>Penicillium chrysogenum</i> (B6HMA9); Af EDP48273, putative STE20-like kinase from <i>Aspergillus fumigatus</i> (BA78_1793), SmKIN3, putative serine/threonine-protein-kinase 3 from <i>S</i>. <i>macrospora</i> (F7VYS5); NcSTK-3 (PRP-9, SID-1) serine/threonine-protein-kinase 3 from <i>N</i>. <i>crassa</i> (V5INC1); Af EDP51073, putative Ste20-like kinase from <i>A</i>. <i>fumigatus</i> (B0Y2A3); Pc21g14960, putative serine/threonine-protein kinase from <i>P</i>. <i>crysogenum</i> (B6HJ11); Fg07344, putative serine/threonine-protein kinase from <i>F</i>. <i>graminearum</i> (A0A016PPL3); SpSID1, serine/threonine-protein kinase from <i>Schizosaccharomyces pombe</i> (O14305); ScKIC1, serine/threonine-protein kinase from <i>Saccharomyces cerevisiae</i> (P38692); SpPPK11, serine/threonine-protein kinase from <i>S</i>. <i>pombe</i> (O14047); ScSPS1, sporulation-specific protein 1 from <i>S</i>. <i>cerevisiae</i> (P08458). Total numbers of amino acids are given in brackets. Regions of the aligned N-terminal domain of the protein sequences are indicated at the end of the alignment. ATP-binding side, catalytic loop, Mg-binding sequence, activation loop and region for substrate binding are marked with red lines. The entire activation segment is underlined in green. Filled box indicate the threonine residue (Thr190 in MST4, Thr182 in STK24, and Thr174 in STK25), phosphorylated during activation.</p

Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the <i>Sordaria macrospora</i> Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex

<div><p>The <u>str</u>iatin-<u>i</u>nteracting <u>p</u>hosphatase <u>a</u>nd <u>k</u>inase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete <i>Sordaria macrospora</i>, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian <u>g</u>erminal <u>c</u>enter <u>k</u>inases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in <i>S</i>. <i>macrospora</i>. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). <i>In vivo</i> localization found that both kinases were present at the septa and deletion of both <i>Smkin3</i> and <i>Smkin24</i> led to abnormal septum distribution. While deletion of <i>Smkin3</i> caused larger distances between adjacent septa and increased aerial hyphae, deletion of <i>Smkin24</i> led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.</p></div

Co-immunoprecipitation of HA-PRO11 and FLAG-SmKIN3 visualized by Western blot analysis.

<p>The strain used for co-IP expressed the respective proteins in suitable amounts (cell extracts); HA-PRO11 shown by anti-HA antibody (anti-HA) and FLAG-SmKIN3 detected by FLAG antibody (anti-FLAG). FLAG-SmKIN3 was pull-downed with HA-PRO11 as bait and identified by Western blot using anti-FLAG antibody (IP-HA). For this experiment, a strain expressing FLAG-SmKIN3 and the HA tag without any fused protein served as negative control and was treated equivalent to the co-IP sample. To exclude unspecific antibody binding, protein extract of the wt was used as second negative control for Western blot analysis. HA-PRO11 was isolated using FLAG-SmKIN3 as bait and identified by Western blot (IP-FLAG) using an anti-HA antibody. A strain expressing genes coding for the FLAG-tag without any fused protein and HA-PRO11 served as negative control and was treated as the co-IP sample. To exclude unspecific antibody binding, protein extract of the wt was used as second negative control for Western blot analysis. “+” mark strains expressing the respective construct.</p

PRO11 interacts physically with SmKIN3 (A) and SmKIN24 (B) in a yeast two-hybrid analysis.

<p>Shown are serial dilutions of diploid yeast strains obtained after mating and spread on SD medium lacking tryptophan (trp) and leucine (leu) or trp, leu and adenine (ade) to verify the interaction of both proteins. The left picture displays a control, which ensures presence of both plasmids in the diploid strain; trp and leu prototrophy are regained by genes present on the plasmid. The right picture displays the interaction assay; ade prototrophy is obtained by positive interaction between the proteins fused to GAL4 binding domain (BD) and GAL4 activation domain (AD). Full length versions of SmKIN3 and SmKIN24 without any introns were tested. GAL4-binding domain was fused to SmKIN3 or SmKIN24 GAL4 activation domain to PRO11. Reverse application of activation and binding domain was not possible due to transactivation of BD-PRO11. Yeast transformants expressing genes coding for BD-SmKIN3 or BD-SmKIN24 and AD-RanBPM served as positive control (46). As negative control a diploid strain carrying empty vectors pGADT7 (AD) and pGBKT7 (BD) was used. No interaction between SmMOB3 and SmKIN3 or SmKIN24 has been observed.</p

Analysis of septal development in wt, ΔSmkin3, ΔSmkin24 and ΔSmkin3/ΔSmkin24 and complemented mutants.

<p>(<b>A</b>) Distribution of septa was investigated under the microscope after 18 h and 32 h past inoculation. Septa were stained with Calcofluor white. Scale bar as indicated. (<b>B</b>) For quantification, distances between adjacent septa were measured over a distance of 18 mm per strain 24 h past inoculation. The total distance of 18 mm was divided into 30 segments of 600 μm. Measurements were binned into five different compartment lengths: 0–30 μm, 31–50 μm, 51–70, 71–100 μm, and more than 100 μm. The number of analyzed compartments was normalized to 100%. Error bars (SD) are given as indicated (n = 30). ΔSmkin3+, ΔSmkin24+ complemented mutants carrying an ectopic copy of the respective wt gene.</p

Localization of SmKIN3-eGFP and SmKIN24-eGFP in <i>S</i>. <i>macrospora</i>.

<p>SmKIN3-eGFP and SmKIN24-eGFP localize to septa. For visualization of cell walls and septa, hyphae were co-stained with Calcofluor white. DIC = differential interference contrast microscopy, scale bars as indicated.</p

Schematic representation of mammalian, <i>D</i>. <i>melanogaster</i> and fungal STRIPAK complex and STRIPAK-like complexes (A) and structure of the Hippo network in animals and the MEN/SIN as well as the RAM/MOR signaling pathway in fungi (B).

<p>(A) Core components of the STRIPAK and STRIPAK-like complex (striatin, Mob3, catalytic and scaffold subunit of the phosphatase PP2A, and STRIP proteins and the respective homologs) are depicted in green, putative homologs of the cortactin-binding protein CTTNBP2 are shown in red, putative homologs of SLMAP in light blue and putative SIKE/FGFR1OP2 in dark blue. STRIPAK GCKs and the associated CCM3 protein are indicated in violet. The arrangements of the subunits in the respective are deduced from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139163#pone.0139163.ref015" target="_blank">15</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139163#pone.0139163.ref020" target="_blank">20</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139163#pone.0139163.ref027" target="_blank">27</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139163#pone.0139163.ref034" target="_blank">34</a>]. (B) Components of the Hippo pathway in <i>D</i>. <i>melanogaster</i> and mammals are indicated in orange and green, respectively. Homologous components of the <i>S</i>. <i>cerevisiae</i> (<i>Sc</i>) MEN and RAM network and the corresponding <i>S</i>. <i>pombe</i> (<i>Sp</i>) SIN and MOR network are shown in the left and right boxes. Components from the filamentous fungus <i>N</i>. <i>crassa</i> (<i>Nc</i>) are shown in red according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139163#pone.0139163.ref035" target="_blank">35</a>]. The figure was modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139163#pone.0139163.ref023" target="_blank">23</a>].</p