Parkinson's disease (PD) is a common neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra and formation of intracellular inclusions known as Lewy bodies in remaining cells. Despite recent development in PD research, rigorous biomarkers for pre-symptomatic diagnosis of PD are not available. Elucidation of the molecular basis of PD pathogenesis will facilitate the search of these biomarkers to allow early treatment. In addition to common sporadic PD, there exist rare familial forms of Parkinsonism, usually characterized by earlier age of onset and, in some cases, absence of typical Lewy body Abbreviations used: HtrA2, high temperature requirement serine protease 2; IKK, IjBa kinase; JNK, c-Jun NH 2 -terminal kinase; NF-jB, nuclear factor-jB; PanK2, pantothenate kinase 2; PD, Parkinson's disease; PI3-kinase, phosphatidylinositol-3 kinase; PINK1, PTEN-induced kinase 1; PTEN, phosphatase and tensin homologue deleted on chromosone 10; PTP, mitochondrial permeability transition pore; ROS, reactive oxygen species; siRNA, small interfering RNA; TIM, translocase of the inner membrane; TRAF, tumor necrosis factor receptor-associated factor; TRAP, tumor necrosis factor receptor-associated protein. AbstractMutations in PTEN-induced kinase 1 (PINK1) gene cause PARK6 familial Parkinsonism. To decipher the role of PINK1 in pathogenesis of Parkinson's disease (PD), researchers need to identify protein substrates of PINK1 kinase activity that govern neuronal survival, and establish whether aberrant regulation and inactivation of PINK1 contribute to both familial Parkinsonism and idiopathic PD. These studies should take into account the several unique structural and functional features of PINK1. First PINK1 is a rare example of a protein kinase with a predicted mitochondrial-targeting sequence and a possible resident mitochondrial function. Second, bioinformatic analysis reveals unique insert regions within the kinase domain that are potentially involved in regulation of kinase activity, substrate selectivity and stability of PINK1. Third, the C-terminal region contains functional motifs governing kinase activity and substrate selectivity. Fourth, accumulating evidence suggests that PINK1 interacts with other signaling proteins implicated in PD pathogenesis and mitochondrial dysfunction. The most prominent examples are the E3 ubiquitin ligase Parkin, the mitochondrial protease high temperature requirement serine protease 2 and the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1. How PINK1 may regulate these proteins to maintain neuronal survival is unclear. This review describes the unique structural features of PINK1 and their possible roles in governing mitochondrial import, processing, kinase activity, substrate selectivity and stability of PINK1. Based upon the findings of previous studies of PINK1 function in cell lines and animal models, we propose a model on the neuroprotective mechanism of PINK1. This model may serve as a conceptual framework...

Background: Depolarization-induced tonic contraction of vascular smooth muscle involves tyrosine phosphorylation. Results: Depolarization activates the Ca 2ϩ -dependent tyrosine kinase Pyk2, leading to activation of the RhoA/Rho-associated kinase pathway. Conclusion: Activation of Pyk2 is required for the sustained phase of depolarization-induced contraction. Significance: Knowledge of the mechanisms responsible for sustained contraction is crucial for identification of defects leading to disease associated with vascular contractile dysfunction.

Various investigators have identified the major domain organization of LRRK2 (leucine-rich repeat kinase 2), which includes a GTPase ROC (Ras of complex proteins) domain followed by a COR (C-terminal of ROC) domain and a protein kinase domain. In addition, there are four domains composed of structural repeat motifs likely to be involved in regulation and localization of this complex protein. In the present paper, we report our bioinformatic analyses of the human LRRK2 amino acid sequence to predict the repeat size, number and likely boundaries for the armadillo repeat, ankyrin repeat, the leucine-rich repeat and WD40 repeat regions of LRRK2. Homology modelling using known protein structures with similar domains was used to predict structures, exposed residues and location of mutations for these repeat regions. We predict that the armadillo repeats, ankyrin repeats and leucine-rich repeats together form an extended N-terminal flexible 'solenoid'-like structure composed of tandem repeat modules likely to be important in anchoring to the membrane and cytoskeletal structures as well as binding to other protein ligands. Near the C-terminus of LRRK2, the WD40 repeat region is predicted to form a closed propeller structure that is important for protein complex formation.

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Genetic variations of leucine-rich repeat kinase 2 (LRRK2) are the major cause of dominantly inherited Parkinson disease (PD). LRRK2 protein contains seven predicted domains: a tandem Ras-like GTPase (ROC) domain and C-terminal of Roc (COR) domain, a protein kinase domain, and four repeat domains. PD-causative variations arise in all domains, suggesting that aberrant functioning of any domain can contribute to neurotoxic mechanisms of LRRK2. Determination of the three-dimensional structure of LRRK2 is one of the best avenues to decipher its neurotoxic mechanism. However, with the exception of the Roc domain, the three-dimensional structures of the functional domains of LRRK2 have yet to be determined. Based on the known three-dimensional structures of repeat domains of other proteins, the tandem Roc-COR domains of the Chlorobium tepidum Rab family protein, and the kinase domain of the Dictyostelium discoideum Roco4 protein, we predicted (1) the motifs essential for protein-protein interactions in all domains, (2) the motifs critical for catalysis and substrate recognition in the tandem Roc-COR and kinase domains, and (3) the effects of some PD-associated missense variations on the neurotoxic action of LRRK2. Results of our analysis provide a conceptual framework for future investigation into the regulation and the neurotoxic mechanism of LRRK2.

Mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene can cause early-onset familial Parkinson disease (PD). PINK1 encodes a neuroprotective protein kinase localized at the mitochondria, and its involvement in regulating mitochondrial dynamics, trafficking, structure, and function is well documented. Owing to the lack of information on structure and biochemical properties for PINK1, exactly how PINK1 exerts its neuroprotective function and how the PD-causative mutations impact on PINK1 structure and function remain unclear. As an approach to address these questions, we conducted bioinformatic analyses of the mitochondrial targeting, the transmembrane, and kinase domains of PINK1 to predict the motifs governing its regulation and function. Our report sheds light on how PINK1 is targeted to the mitochondria and how PINK1 is cleaved by mitochondrial peptidases. Moreover, it includes a potential optimal phosphorylation sequence preferred by the PINK1 kinase domain. On the basis of the results of our analyses, we predict how the PD-causative mutations affect processing of PINK1 in the mitochondria, PINK1 kinase activity, and substrate specificity. In summary, our results provide a conceptual framework for future investigation of the structural and biochemical basis of regulation and the neuroprotective mechanism of PINK1.

C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) are endogenous inhibitors constraining the activity of the oncogenic Src-family kinases (SFKs) in cells. Both kinases suppress SFKs by selectively phosphorylating their consensus C-terminal regulatory tyrosine. In addition to phosphorylation, CHK can suppress SFKs by a unique non-catalytic inhibitory mechanism that involves tight binding of CHK to SFKs to form stable complexes. In this review, we discuss how allosteric regulators, phosphorylation, and inter-domain interactions interplay to govern the activity of CSK and CHK and their ability to inhibit SFKs. In particular, based upon the published results of structural and biochemical analysis of CSK and CHK, we attempt to chart the allosteric networks in CSK and CHK that govern their catalysis and ability to inhibit SFKs. We also discuss how the published three-dimensional structure of CSK complexed with an SFK member sheds light on the structural basis of substrate recognition by protein kinases.

1. The Src-family protein tyrosine kinases (SFKs) are multidomain oncogenic protein tyrosine kinases. Their overactivation contributes to cancer formation and progression. Thus, synthetic inhibitors of SFKs are being developed as therapeutics for cancer treatment. Understanding the regulatory and catalytic mechanisms of SFKs is necessary for the development of therapeutic SFK inhibitors. 2. Although many upstream regulators and protein substrates of SFKs have been identified, both the mechanisms of activation and catalysis of SFKs are not fully understood. In particular, it is still unclear how the inactive SFKs undergo conformational transition during activation. The mechanism governing the binding of substrates and the release of products during catalysis is another area that requires investigation. 3. Several recent publications indicate the presence of a 'hydrophobic spine' formed by four conserved interacting hydrophobic residues in the kinase domain of SFKs. In the present review, we discuss how the assembly and disassembly of the hydrophobic spine residues may govern conformational transition of SFKs during activation. In addition to regulation of kinase activity, the hydrophobic spine is implicated to be involved in catalysis. It has been postulated recently that perturbation of the hydrophobic spine residues is a key step in catalysis. 4. Further investigations to decipher the roles of the hydrophobic spine residues in regulation and catalysis of SFKs will benefit the development of therapeutic SFK inhibitors for cancer treatment.