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

Frey S, Reschka EJ, Pöggeler S - PLoS ONE (2015)

Bottom Line: Here, we report on the presence and function of STRIPAK-associated kinases in 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).In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August-University Göttingen, Göttingen, Germany.

ABSTRACT
The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, 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 germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. 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). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 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.

No MeSH data available.


Related in: MedlinePlus

PRO11 interacts physically with SmKIN3 (A) and SmKIN24 (B) in a yeast two-hybrid analysis.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.
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pone.0139163.g004: PRO11 interacts physically with SmKIN3 (A) and SmKIN24 (B) in a yeast two-hybrid analysis.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.

Mentions: Striatin is the scaffold of mammalian STRIPAK complex kinases MST4, STK24, STK25 and MINK1 [15, 16]. PRO11 was previously shown to be the S. macrospora homolog of mammalian striatin [8]. The sequence similarity between SmKIN3 and SmKIN24 and the mammalian kinases of the GCK III and GCK IV family (Fig 1) led us to inspect whether both S. macrospora kinases can interact with PRO11 in a Y2H system. Full-length cDNAs (version I with all introns spliced, Fig A in S1 File) of Smkin3 and Smkin24 were cloned into the Y2H vector pGBKT7 that contains the GAL4 DNA-binding domain. The pGADT7-derived plasmid pAD11FL, which encodes a full-length PRO11 fused to the GAL4 activation domain, served as the prey vector. Plasmids were transformed into yeast strains Y187 (pGBKT7 constructs) or AH109 (pGADT7 constructs). Strains carrying pBD-SmKIN3 and pBD-SmKIN24 plasmids were checked for transactivation activity by mating with yeast strain AH109 containing pGADT7. A strain carrying pGBKT7 and pGADT7 was used as negative control. Gal4 fusion proteins of SmKIN3 and SmKIN24 were checked for adequate expression and two-hybrid competency using the RanBPM system [62]. Previously, we showed that the MOB domain protein SmMOB3, the mammalian phocein homologue, is a strong interaction partner of PRO11 [31, 74]. We therefore tested the interaction of both kinases with SmMOB3. Using the Y2H system we confirmed that both SmKIN3 and SmKIN24 could physically interact with PRO11 but not with SmMOB3 (Fig 4 and Fig D in S1 File).


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

Frey S, Reschka EJ, Pöggeler S - PLoS ONE (2015)

PRO11 interacts physically with SmKIN3 (A) and SmKIN24 (B) in a yeast two-hybrid analysis.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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4587736&req=5

pone.0139163.g004: PRO11 interacts physically with SmKIN3 (A) and SmKIN24 (B) in a yeast two-hybrid analysis.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.
Mentions: Striatin is the scaffold of mammalian STRIPAK complex kinases MST4, STK24, STK25 and MINK1 [15, 16]. PRO11 was previously shown to be the S. macrospora homolog of mammalian striatin [8]. The sequence similarity between SmKIN3 and SmKIN24 and the mammalian kinases of the GCK III and GCK IV family (Fig 1) led us to inspect whether both S. macrospora kinases can interact with PRO11 in a Y2H system. Full-length cDNAs (version I with all introns spliced, Fig A in S1 File) of Smkin3 and Smkin24 were cloned into the Y2H vector pGBKT7 that contains the GAL4 DNA-binding domain. The pGADT7-derived plasmid pAD11FL, which encodes a full-length PRO11 fused to the GAL4 activation domain, served as the prey vector. Plasmids were transformed into yeast strains Y187 (pGBKT7 constructs) or AH109 (pGADT7 constructs). Strains carrying pBD-SmKIN3 and pBD-SmKIN24 plasmids were checked for transactivation activity by mating with yeast strain AH109 containing pGADT7. A strain carrying pGBKT7 and pGADT7 was used as negative control. Gal4 fusion proteins of SmKIN3 and SmKIN24 were checked for adequate expression and two-hybrid competency using the RanBPM system [62]. Previously, we showed that the MOB domain protein SmMOB3, the mammalian phocein homologue, is a strong interaction partner of PRO11 [31, 74]. We therefore tested the interaction of both kinases with SmMOB3. Using the Y2H system we confirmed that both SmKIN3 and SmKIN24 could physically interact with PRO11 but not with SmMOB3 (Fig 4 and Fig D in S1 File).

Bottom Line: Here, we report on the presence and function of STRIPAK-associated kinases in 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).In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August-University Göttingen, Göttingen, Germany.

ABSTRACT
The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, 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 germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. 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). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 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.

No MeSH data available.


Related in: MedlinePlus