Biomedical subjects

Ruben K Dagda

Publications and source records attributed to Ruben K Dagda.

3 recordsLinked to original sources

PubMed2005

Unfolding-resistant translocase targeting: a novel mechanism for outer mitochondrial membrane localization exemplified by the Bbeta2 regulatory subunit of protein phosphatase 2A.

Heterotrimeric serine/threonine protein phosphatase 2A (PP2A) consists of scaffolding (A), catalytic (C), and variable (B, B', and B'') subunits. Variable subunits dictate subcellular localization and substrate specificity of the PP2A holoenzyme. The Bbeta regulatory subunit gene is mutated in spinocerebellar ataxia type 12, and one of its splice variants, Bbeta2, targets PP2A to mitochondria to promote apoptosis in PC12 cells (Dagda, R. K., Zaucha, J. A., Wadzinski, B. E., and Strack, S. (2003) J. Biol. Chem. 278, 24976-24985). Here, we report that Bbeta2 is localized to the outer mitochondrial membrane by a novel mechanism, combining a cryptic mitochondrial import signal with a structural arrest domain. Scanning mutagenesis demonstrates that basic and hydrophobic residues mediate mitochondrial association and the proapoptotic activity of Bbeta2. When fused to green fluorescent protein, the N terminus of Bbeta2 acts as a cleavable mitochondrial import signal. Surprisingly, full-length Bbeta2 is not detectably cleaved and is retained at the outer mitochondrial membrane, even though it interacts with the TOM22 import receptor, as shown by luciferase complementation in intact cells. Mutations that open the C-terminal beta-propeller of Bbeta2 facilitate mitochondrial import, indicating that this rigid fold acts as a stop-transfer domain by resisting the partial unfolding step prerequisite for matrix translocation. Because hybrids of prototypical import and beta-propeller domains recapitulate this behavior, we predict the existence of other similarly localized proteins and a selection against highly stable protein folds in the mitochondrial matrix. This unfolding-resistant targeting to the mitochondrial translocase is necessary but not sufficient for the proapoptotic activity of Bbeta2, which also requires association with the rest of the PP2A holoenzyme.

Animals↗

PubMed2003

A developmentally regulated, neuron-specific splice variant of the variable subunit Bbeta targets protein phosphatase 2A to mitochondria and modulates apoptosis.

Heterotrimeric protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase composed of catalytic, structural, and regulatory subunits. Here, we characterize Bbeta2, a novel splice variant of the neuronal Bbeta regulatory subunit with a unique N-terminal tail. Bbeta2 is expressed predominantly in forebrain areas, and PP2A holoenzymes containing Bbeta2 are about 10-fold less abundant than those containing the Bbeta1 (previously Bbeta) isoform. Bbeta2 mRNA is dramatically induced postnatally and in response to neuronal differentiation of a hippocampal progenitor cell line. The divergent N terminus of Bbeta2 does not affect phosphatase activity but encodes a subcellular targeting signal. Bbeta2, but not Bbeta1 or an N-terminal truncation mutant, colocalizes with mitochondria in neuronal PC12 cells. Moreover, the Bbeta2 N-terminal tail is sufficient to target green fluorescent protein to this organelle. Inducible or transient expression of Bbeta2, but neither Bbeta1, Bgamma, nor a Bbeta2 mutant defective in holoenzyme formation, accelerates apoptosis in response to growth factor deprivation. Thus, alternative splicing of a mitochondrial localization signal generates a PP2A holoenzyme involved in neuronal survival signaling.

Alternative Splicing↗

PubMed2002

Protein phosphatase 2A holoenzyme assembly: identification of contacts between B-family regulatory and scaffolding A subunits.

Protein serine/threonine phosphatase (PP) 2A is a ubiquitous enzyme with pleiotropic functions. Trimeric PP2A consists of a structural A subunit, a catalytic C subunit, and a variable regulatory subunit. Variable subunits (B, B', and B" families) dictate PP2A substrate specificity and subcellular localization. B-family subunits contain seven WD repeats predicted to fold into a beta-propeller structure. We carried out mutagenesis of Bgamma to identify domains important for association with A and C subunits in vivo. Several internal deletions in Bgamma abolished coimmunoprecipitation of A and C subunits expressed in COS-M6 cells. In contrast, small N- and C-terminal Bgamma deletions had no effect on incorporation into the PP2A heterotrimer. Thus, holoenzyme association of B-family subunits requires multiple, precisely aligned contacts within a core beta-propeller domain. Charge-reversal mutagenesis of Bgamma identified a cluster of conserved critical residues in Bgamma WD repeats 3 and 4. Acidic substitution of paired basic residues in Bgamma (RR165EE) abolished association with wild-type A and C subunits, while fostering incorporation of Bgamma into a PP2A heterotrimer containing an A subunit with an opposite charge-reversal mutation (EE100RR). Thus, binding of A and B subunits requires electrostatic interactions between conserved pairs of glutamates and arginines. By expressing complementary charge-reversal mutants in neuronal PC6-3 cells, we further show that holoenzyme incorporation protects Bgamma from rapid degradation by the ubiquitin/proteasome pathway.

Amino Acid Sequence↗