Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and idiopathic Parkinson's disease. However, the mechanisms for activating its physiological function are not known, hindering identification of the biological role of endogenous LRRK2. The recent discovery that LRRK2 is highly expressed in cells of the innate immune system and genetic association is a risk factor for autoimmune disorders implies an important role for LRRK2 in pathology outside of the central nervous system. Thus, an examination of endogenous LRRK2 in immune cells could provide insight into the protein's function. Here, we establish that stimulation of specific Toll-like receptors results in a complex biochemical activation of endogenous LRRK2, with early phosphorylation of LRRK2 preceding its dimerization and membrane translocation. Membrane-associated LRRK2 co-localized to autophagosome membranes following either TLR4 stimulation or mTOR inhibition with rapamycin. Silencing of endogenous LRRK2 expression resulted in deficits in the induction of autophagy and clearance of a well-described macroautophagy substrate, demonstrating the critical role of endogenous LRRK2 in regulating autophagy. Inhibition of LRRK2 kinase activity also reduced autophagic degradation and suggested the importance of the kinase domain in the regulation of autophagy. Our results demonstrate a well-orchestrated series of biochemical events involved in the activation of LRRK2 important to its physiological function. With similarities observed across multiple cell types and stimuli, these findings are likely relevant in all cell types that natively express endogenous LRRK2, and provide insights into LRRK2 function and its role in human disease.

Phosphorylation is the most common and pleiotropic modification in biology, which plays a vital role in regulating and finely tuning a multitude of biological pathways. Transport across the nuclear envelope is also an essential cellular function and is intimately linked to many degeneration processes that lead to disease. It is therefore not surprising that phosphorylation of cargos trafficking between the cytoplasm and nucleus is emerging as an important step to regulate nuclear availability, which directly affects gene expression, cell growth and proliferation. However, the literature on phosphorylation of nucleocytoplasmic trafficking cargos is often confusing. Phosphorylation, and its mirror process dephosphorylation, has been shown to have opposite and often contradictory effects on the ability of cargos to be transported across the nuclear envelope. Without a clear connection between attachment of a phosphate moiety and biological response, it is difficult to fully understand and predict how phosphorylation regulates nucleocytoplasmic trafficking. In this review, we will recapitulate clue findings in the field and provide some general rules on how reversible phosphorylation can affect the nuclear-cytoplasmic localization of substrates. This is only now beginning to emerge as a key regulatory step in biology.

Missense mutations in the multi-domain kinase LRRK2 cause late onset familial Parkinson's disease. They most commonly with classic proteinopathy in the form of Lewy bodies and Lewy neurites comprised of insoluble α-synuclein, but in rare cases can also manifest tauopathy. The normal function of LRRK2 has remained elusive, as have the cellular consequences of its mutation. Data from LRRK2 null model organisms and LRRK2-inhibitor treated animals support a physiological role for LRRK2 in regulating lysosome function. Since idiopathic and LRRK2-linked PD are associated with the intraneuronal accumulation of protein aggregates, a series of critical questions emerge. First, how do pathogenic mutations that increase LRRK2 kinase activity affect lysosome biology in neurons? Second, are mutation-induced changes in lysosome function sufficient to alter the metabolism of α-synuclein? Lastly, are changes caused by pathogenic mutation sensitive to reversal with LRRK2 kinase inhibitors? Here, we report that mutation of LRRK2 induces modest but significant changes in lysosomal morphology and acidification, and decreased basal autophagic flux when compared to WT neurons. These changes were associated with an accumulation of detergent-insoluble α-synuclein and increased neuronal release of α-synuclein and were reversed by pharmacologic inhibition of LRRK2 kinase activity. These data demonstrate a critical and disease-relevant influence of native neuronal LRRK2 kinase activity on lysosome function and α-synuclein homeostasis. Furthermore, they also suggest that lysosome dysfunction, altered neuronal α-synuclein metabolism, and the insidious accumulation of aggregated protein over decades may contribute to pathogenesis in this late-onset form of familial PD.

The proteins alpha-synuclein (αSyn) and LRRK2 are both key players in the pathogenesis of the neurodegenerative disorder Parkinson’s disease (PD), but establishing a functional link between the two proteins has proven elusive. Research studies for these two proteins have traditionally and justifiably focused in neuronal cells, but recent studies indicate that each protein could play a greater pathological role elsewhere. αSyn is expressed at high levels within neurons, but they also secrete the protein into the extracellular milieu, where it can have broad ranging effects in the nervous system and relevance to disease etiology. Similarly, low neuronal LRRK2 expression and activity suggests that LRRK2-related functions could be more relevant in cells with higher expression, such as brain-resident microglia. Microglia are monocytic immune cells that protect neurons from noxious stimuli, including pathological αSyn species, and microglial activation is believed to contribute to neuroinflammation and neuronal death in PD. Interestingly, both αSyn and LRRK2 can be linked to microglial function. Secreted αSyn can directly activate microglia, and can be taken up by microglia for clearance, while LRRK2 has been implicated in the intrinsic regulation of microglial activation and of lysosomal degradation processes. Based on these observations, the present review will focus on how PD-associated mutations in LRRK2 could potentially alter microglial biology with respect to neuronally-secreted αSyn, resulting in cell dysfunction and neurodegeneration.

The Vaccinia virus H1 gene product, VH1, is a dual specificity phosphatase that down-regulates the cellular antiviral response by dephosphorylating STAT1. The crystal structure of VH1, determined at 1.32 Å resolution, reveals a novel dimeric quaternary structure, which exposes two active sites spaced ϳ39 Å away from each other. VH1 forms a stable dimer via an extensive domain swap of the N-terminal helix (residues 1-20). In vitro, VH1 can dephosphorylate activated STAT1, in a reaction that is competed by the nuclear transport adapter importin ␣5. Interestingly, VH1 is inactive with respect to STAT1 bound to DNA, suggesting that the viral phosphatase acts predominantly on the cytoplasmic pool of activated STAT1. We propose that the dimeric quaternary structure of VH1 is essential for specific recognition of activated STAT1, which prevents its nuclear translocation, thus blocking interferon-␥ signal transduction and antiviral response. Dual specificity phosphates (DSPs)2 comprise a growing subclass of protein-tyrosine phosphatases, which dephosphorylate both phosphotyrosine and phosphoserine/threonine residues. The first identified DSP, VH1, is the product of the Vaccinia virus gene H1 (1). To date, the small VH1 (ϳ20 kDa) is the prototype of a family of VH1-like DSPs found in plants, yeast, insects, and higher eukaryotes (2). The human genome encodes at least 38 VH1-like phosphatases, which regulate many critical aspects of the cell cycle (3). VH1-like DSPs share a common catalytic mechanism, which is mediated by a catalytic triad consisting of a cysteine, an arginine, and an aspartic acid, usually present in the context of an extended consensus motif (4). The structural organization of the minimum catalytic core of VH1-like DSPs is known from the crystal structures of several members of the VH1-like family, such as VHZ (5) and VHR (6). All known DSPs share a common topology with members of the classical protein-tyrosine phosphatases, with the most marked structural difference being in the architecture of the active site. To accommodate both phosphotyrosine and phosphothreonine/serine residues, DSPs present a shallow catalytic cleft only ϳ6 Å deep. In contrast, the catalytic cysteine residue of classical protein-tyrosine phosphatases sits at the bottom of a ϳ9-Å-deep pocket, which selectively recognizes bulky phosphotyrosines (6). In vitro, VH1 and many other VH1-like DSPs are characterized by resistance to okadaic acid and sensitivity to sodium vanadate (1). Sodium vanadate acts as a potent inhibitor of cysteine-phosphatases by covalently labeling the cysteine group in the active site (4).The gene encoding VH1 is highly conserved among poxviruses and essential for the viability of Vaccinia virus in tissue cultures (7). VH1 is expressed in the late stage of viral infection, and ϳ200 molecules of VH1 are packaged within the virion (7). The conservation of the VH1 gene in poxviruses as well as its essential role for virus viability emphasize VH1 involvement in a critical step of the virus life cycle. Recent evidenc...

Interferon-γ (IFN-γ) stimulation triggers tyrosine-phosphorylation of transcription factor STAT1 at position 701, which is associated with the switching from carrier-independent nucleocytoplasmic shuttling to carrier-mediated nuclear import. Unlike most substrates that carry a classical Nuclear Localization Signal (cNLS) and bind to importin α1, STAT1 possesses a nonclassical NLS recognized by the isoform importin α5. In the present study, we have analyzed the mechanisms by which importin α5 binds phosphorylated STAT1 (pSTAT1). We found that a homodimer of pSTAT1 is recognized by one equivalent of importin α5 with K d = 191 ± 20 nM. Whereas tyrosine-phosphorylation at position 701 is essential to assemble a pSTAT1:importin α5 complex, the phosphate moiety is not a direct binding determinant for importin α5. In contrast to classical NLS substrates, pSTAT1 binding to importin α5 is not displaced by the N-terminal importin β binding (IBB)-domain, and requires the importin α5 C-terminal acidic tail (505-EEDD-508). A local unfolding of importin α5 ARM repeat 10 accompanies high affinity binding to pSTAT1. This unfolding is mediated by a single conserved tyrosine at position 476 of importin α5, which is inserted in between Armadillo (ARM) repeat 10 helices H1-H2-H3, thereby preventing the intramolecular helical stacking essential to stabilize the folding conformation of ARM 10. Introducing a glycine at this position, as in importin α1, disrupts high-affinity binding to pSTAT1, suggesting pSTAT1 recognition is dependent on the intrinsic flexibility of ARM 10. Using the quantitative stoichiometry and binding data presented in this paper together with mutational information available in the literature, we propose importin α5 binds in between two STAT1 monomers, with two major binding determinants in the SH2-and DNA-binding domains. In vitro this model is supported by the observation that a 38mer DNA oligonucleotide containing two tandem cfosM67-promoters can displace importin α5 from pSTAT1, suggesting a possible role for DNA in releasing activated STAT1 in the cell nucleus.

Background:The mechanisms of homologous recombination and recombinational repair of double-stranded DNA breaks in mitochondria are poorly understood. Results: The yeast mitochondrial nucleoid protein, Mgm101, shares biochemical, structural, and functional similarities with the Rad52 family proteins. Conclusion: Mgm101 is a Rad52-related molecular component involved in mtDNA recombination Significance: This finding helps in understanding the mechanism of homologous recombination in mitochondria.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder, affecting 1–3% of the population over 65. Mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive PD. The parkin protein possesses potent cell-protective properties and has been mechanistically linked to both the regulation of apoptosis and the turnover of damaged mitochondria. Here, we explored these two functions of parkin and the relative scale of these processes in various cell types. While biochemical analyses and subcellular fractionation were sufficient to observe robust parkin-dependent mitophagy in immortalized cells, higher resolution techniques appear to be required for primary culture systems. These approaches, however, did affirm a critical role for parkin in the regulation of apoptosis in primary cultured neurons and all other cells studied. Our prior work demonstrated that parkin-dependent ubiquitination of endogenous Bax inhibits its mitochondrial translocation and can account for the anti-apoptotic effects of parkin. Having found a central role for parkin in the regulation of apoptosis, we further investigated the parkin-Bax interaction. We observed that the BH3 domain of Bax is critical for its recognition by parkin, and identified two lysines that are crucial for parkin-dependent regulation of Bax translocation. Last, a disease-linked mutation in parkin failed to influence Bax translocation to mitochondria after apoptotic stress. Taken together, our data suggest that regulation of apoptosis by the inhibition of Bax translocation is a prevalent physiological function of parkin regardless of the kind of cell stress, preventing overt cell death and supporting cell viability during mitochondrial injury and repair.