Abstract
The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants (Δpro11, Δpro11Δpro22, Δpp2Ac1Δpro22) from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues. While S180 was dephosphorylated in a STRIPAK-dependent manner, S216, and S1343 served as non-regulated phosphorylation sites. While the S1343 mutants were indistinguishable from wild type, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth, and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microscopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -independent phosphorylation of GUL1 regulates sexual development and asexual growth.
Highlights
Protein phosphatase PP2A is the most abundant phosphatase in eukaryotes
The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex controls a variety of developmental processes, and the lack of single STRIPAK subunits is associated with severe developmental defects and diseases
The filamentous fungus Sordaria macrospora is a well-established model system used to study the function of STRIPAK, since a collection of STRIPAK mutants is experimentally accessible
Summary
Protein phosphatase PP2A is the most abundant phosphatase in eukaryotes. An affinity purification/mass spectrometry approach using human cells identified a novel large multiprotein assembly centered on PP2A and was called the “striatin-interacting phosphatase and kinase” (STRIPAK) multi-subunit complex. Besides A and C subunits of PP2A, it contains the regulatory B”‘ subunit striatin [3]. STRIPAK complexes are highly conserved within eukaryotes, and have been detected in diverse animal tissues, as well as in filamentous fungi [4, 5]. Further constituents of the STRIPAK core complex include the striatin-interacting proteins STRIP1/2, Mob3/phocein, cerebral cavernous malformation 3 (CCM3), and two associated subunits, the sarcolemmal membrane-associated protein (SLMAP), and the coiled-coil protein suppressor of IκB kinase-ε (IKKε) designated as SIKE [6]. The phosphorylation activity of STRIPAK is dependent on germinal center kinases (GCKs)
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