Protein phosphatase 2A is crucial for sarcomere organization in Caenorhabditis elegans striated muscle.

Protein phosphatase 2A (PP2A) is a heterotrimer composed of single catalytic and scaffolding subunits and one of several possible regulatory subunits. We identified PPTR-2, a regulatory subunit of PP2A, as a binding partner for the giant muscle protein UNC-89 (obscurin) in Caenorhabditis elegans. PPTR-2 is required for sarcomere organization when its paralogue, PPTR-1, is deficient. PPTR-2 localizes to the sarcomere at dense bodies and M-lines, colocalizing with UNC-89 at M-lines. PP2A components in C. elegans include one catalytic subunit LET-92, one scaffolding subunit (PAA-1), and five regulatory subunits (SUR-6, PPTR-1, PPTR-2, RSA-1, and CASH-1). In adult muscle, loss of function in any of these subunits results in sarcomere disorganization. rsa-1 mutants show an interesting phenotype: one of the two myosin heavy chains, MHC A, localizes as closely spaced double lines rather than single lines. This "double line" phenotype is found in rare missense mutants of the head domain of MHC B myosin, such as unc-54(s74). Analysis of phosphoproteins in the unc-54(s74) mutant revealed two additional phosphoserines in the nonhelical tailpiece of MHC A. Antibodies localize PPTR-1, PAA-1, and SUR-6 to I-bands and RSA-1 to M-lines and I-bands. Therefore, PP2A localizes to sarcomeres and functions in the assembly or maintenance of sarcomeres.

Protein phosphatase 2a (PP2A) binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3.

BACKGROUND: Striatin, a putative protein phosphatase 2A (PP2A) B-type regulatory subunit, is a multi-domain scaffolding protein that has recently been linked to several diseases including cerebral cavernous malformation (CCM), which causes symptoms ranging from headaches to stroke. Striatin association with the PP2A A/C (structural subunit/catalytic subunit) heterodimer alters PP2A substrate specificity, but targets and roles of striatin-associated PP2A are not known. In addition to binding the PP2A A/C heterodimer to form a PP2A holoenzyme, striatin associates with cerebral cavernous malformation 3 (CCM3) protein, the mammalian Mps one binder (MOB) homolog, Mob3/phocein, the mammalian sterile 20-like (Mst) kinases, Mst3, Mst4 and STK25, and several other proteins to form a large signaling complex. Little is known about the molecular architecture of the striatin complex and the regulation of these sterile 20-like kinases. RESULTS: To help define the molecular organization of striatin complexes and to determine whether Mst3 might be negatively regulated by striatin-associated PP2A, a structure-function analysis of striatin was performed. Two distinct regions of striatin are capable of stably binding directly or indirectly to Mob3--one N-terminal, including the coiled-coil domain, and another more C-terminal, including the WD-repeat domain. In addition, striatin residues 191-344 contain determinants necessary for efficient association of Mst3, Mst4, and CCM3. PP2A associates with the coiled-coil domain of striatin, but unlike Mob3 and Mst3, its binding appears to require striatin oligomerization. Deletion of the caveolin-binding domain on striatin abolishes striatin family oligomerization and PP2A binding. Point mutations in striatin that disrupt PP2A association cause hyperphosphorylation and activation of striatin-associated Mst3. CONCLUSIONS: Striatin orchestrates the regulation of Mst3 by PP2A. It binds Mst3 likely as a dimer with CCM3 via residues lying between striatin's calmodulin-binding and WD-domains and recruits the PP2A A/C heterodimer to its coiled-coil/oligomerization domain. Residues outside the previously reported coiled-coil domain of striatin are necessary for its oligomerization. Striatin-associated PP2A is critical for Mst3 dephosphorylation and inactivation. Upon inhibition of PP2A, Mst3 activation appears to involve autophosphorylation of multiple activation loop phosphorylation sites. Mob3 can associate with striatin sequences C-terminal to the Mst3 binding site but also with sequences proximal to striatin-associated PP2A, consistent with a possible role for Mob 3 in the regulation of Mst3 by PP2A.

Genomic organization of RNA2 of Tomato ringspot virus: processing at a third cleavage site in the N-terminal region of the polyprotein in vitro.

The proteinase of Tomato ringspot virus (genus Nepovirus) is responsible for proteolytic cleavage of the RNA2-encoded polyprotein (P2) at two cleavage sites, allowing definition of the domains for the movement protein (MP) and coat protein. In this study, we have characterized a third cleavage site in the N-terminal region of P2 using an in vitro processing assay and partial cDNA clones. Results from site-directed mutagenesis of putative cleavage sites suggest that cleavage occurs at dipeptide Q(301)/G. Cleavage at this site is predicted to result in the release of two proteins from the N-terminal region of P2: a 34 kDa protein located at the N terminus of P2 (assuming translation initiation at the first AUG codon) and a 71 kDa protein located immediately upstream of the MP domain. In contrast, only one protein domain is present in the equivalent region of the P2 polyprotein of other characterized nepoviruses.