Combined kinase inhibition modulates parkin inactivation.

Mutations in the parkin gene cause autosomal-recessive, juvenile-onset parkinsonism, and parkin dysfunction may also play a role in the pathogenesis of sporadic Parkinson disease (PD). Although its precise function remains largely unknown, parkin seems to play a neuroprotective role. Several studies indicate that changes in parkin solubility induced by post-translational modifications, such as S-nitrosylation or dopamine modification, comprise one mechanism of parkin inactivation associated with disease. Protein phosphorylation events have recently been linked to the molecular mechanism(s) underlying PD, but the role of this post-translational modification for parkin function has remained unclear. Here we report that compound phosphorylation of parkin by both casein kinase I and cyclin-dependent kinase 5 (cdk5) decreases parkin solubility, leading to its aggregation and inactivation. Combined kinase inhibition partially reverses the aggregative properties of several pathogenic point mutants in cultured cells. Enhanced parkin phosphorylation is detected in distinct brain areas of individuals with sporadic PD and correlates with increases in the levels of p25, the activator of cdk5. These findings indicate that casein kinase I and cdk5 may represent novel combinatorial therapeutic targets for treating PD.

Modifications of a Nanomolar Cyclic Peptide Antagonist for the EphA4 Receptor To Achieve High Plasma Stability.

EphA4 is a receptor tyrosine kinase with a critical role in repulsive axon guidance and synaptic function. However, aberrant EphA4 activity can inhibit neural repair after injury and exacerbate neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's. We previously identified the cyclic peptide APY-d2 (APYCVYRßASWSC-nh2, containing a disulfide bond) as a potent and selective EphA4 antagonist. However, APY-d2 lacks sufficient plasma stability to be useful for EphA4 inhibition in vivo through peripheral administration. Using structure-activity relationship studies, we show that protecting the peptide N-terminus from proteolytic degradation dramatically increases the persistence of the active peptide in plasma and that a positively charged peptide N-terminus is essential for high EphA4 binding affinity. Among several improved APY-d2 derivatives, the cyclic peptides APY-d3 (ßAPYCVYRßASWSC-nh2) and APY-d4 (ßAPYCVYRßAEWEC-nh2) combine high stability in plasma and cerebrospinal fluid with slightly enhanced potency. These properties make them valuable research tools and leads toward development of therapeutics for neurological diseases.

Attenuation of eph receptor kinase activation in cancer cells by coexpressed ephrin ligands.

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.

Profiling Eph receptor expression in cells and tissues: a targeted mass spectrometry approach.

The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression.

Mechanistic insight into the dominant mode of the Parkinson's disease-associated G2019S LRRK2 mutation.

Pathogenic mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant and certain cases of sporadic Parkinson's disease (PD). The G2019S substitution in LRRK2 is the most common genetic determinant of PD identified so far, and maps to a specific region of the kinase domain called the activation segment. Here, we show that autophosphorylation of LRRK2 is an intermolecular reaction and targets two residues within the activation segment. The prominent pathogenic G2019S mutation in LRRK2 results in altered autophosphorylation, and increased autophosphorylation and substrate phosphorylation, through a process that seems to involve reorganization of the activation segment. Our results suggest a molecular mechanistic explanation for how the G2019S mutation enhances the catalytic activity of LRRK2, thereby leading to pathogenicity. These findings have important implications for therapeutic strategies in PD.