Biology subjects

Clark, K. C.

Publications and source records attributed to Clark, K. C..

3 recordsLinked to original sources

bioRxiv2024

Feed-forward stimulation of CAMK2 by the oncogenic pseudokinase PEAK1 generates a therapeutically "actionable" signalling axis in triple negative breast cancer

The PEAK family of pseudokinases, comprising PEAK1-3, are signalling scaffolds that play oncogenic roles in several poor prognosis human cancers, including triple negative breast cancer (TNBC). However, therapeutic targeting of pseudokinases is challenging due to their lack of catalytic activity. To address this, we screened for PEAK1 effectors by affinity purification and mass spectrometry, identifying calcium/calmodulin-dependent protein kinase 2 (CAMK2)D and CAMK2G. PEAK1 promoted CAMK2D/G activation in TNBC cells via a novel feed-forward mechanism involving PEAK1/PLC{gamma}1/Ca2+ signalling and direct binding via a consensus CAMK2 interaction motif in the PEAK1 N-terminus. In turn, CAMK2 phosphorylated PEAK1 to enhance association with PEAK2, which is critical for PEAK1 oncogenic signalling. To achieve pharmacologic targeting of PEAK1/CAMK2, we repurposed RA306, a second generation CAMK2 inhibitor under pre-clinical development for treatment of cardiovascular disease. RA306 demonstrated on-target activity against CAMK2 in TNBC cells and inhibited PEAK1-enhanced migration and invasion in vitro. Moreover, RA306 significantly attenuated TNBC xenograft growth and blocked metastasis in a manner mirrored by CRISPR-mediated PEAK1 ablation. Overall, these studies establish PEAK1 as a critical cell signalling nexus, identify a novel mechanism for regulation of Ca2+ signalling and its integration with tyrosine kinase signals, and identify CAMK2 as a therapeutically actionable target downstream of PEAK1.

cancer biology↗

bioRxiv2022

Deficiency in the cell-adhesion molecule dscaml1 impairs hypothalamic CRH neuron development and neuroendocrine stress axis function

The corticotropin-releasing hormone (CRH)-expressing neurons in the hypothalamus are critical regulators of the neuroendocrine stress response pathway, known as the hypothalamic-pituitary-adrenal (HPA) axis. As developmental vulnerabilities of CRH neurons contribute to stress-associated neurological and behavioral dysfunctions, it is critical to identify the mechanisms underlying normal and abnormal CRH neuron development. Using zebrafish, we identified Down syndrome cell adhesion molecule like-1 (dscaml1) as an integral mediator of CRH neuron development and necessary for establishing normal stress axis function. In dscaml1 mutant animals, hypothalamic CRH neurons had higher crhb (the CRH homolog in fish) expression, increased cell number, and reduced cell death compared to wild-type controls. Physiologically, dscaml1 mutant animals had higher baseline stress hormone (cortisol) levels and attenuated responses to acute stressors. Together, these findings identify dscaml1 as an essential factor for stress axis development and suggest that HPA axis dysregulation may contribute to the etiology of human DSCAML1 -linked neuropsychiatric disorders.

developmental biology↗

bioRxiv2020

Effects of constitutive and acute Connexin 36 deficiency on brain-wide susceptibility to PTZ-induced neuronal hyperactivity

Connexins are transmembrane proteins that form hemichannels allowing the exchange of molecules between the extracellular space and cell interior. Two hemichannels from adjacent cells dock and form a continuous gap junction pore, thereby permitting direct intercellular communication. Connexin 36 (Cx36), expressed primarily in neurons, is involved in the synchronous activity of neurons and may play a role in aberrant synchronous firing, as seen in seizures. To understand the reciprocal interactions between Cx36 and seizure-like neural activity, we examined three questions: a) does Cx36 deficiency affect seizure susceptibility, b) does seizure-like activity affect Cx36 expression patterns, and c) does acute blockade of Cx36 conductance increase seizure susceptibility. We utilize the zebrafish pentylenetetrazol (PTZ; a GABA(A) receptor antagonist) induced seizure model, taking advantage of the compact size and optical translucency of the larval zebrafish brain to assess how PTZ affects brain-wide neuronal activity and Cx36 protein expression. We exposed wild-type and genetic Cx36-deficient (cx35.5- /-) zebrafish larvae to PTZ and subsequently mapped neuronal activity across the whole brain, using phosphorylated extracellular-signal-regulated kinase (pERK) as a proxy for neuronal activity. We found that cx35.5-/- fish exhibited region-specific susceptibility and resistance to PTZ-induced hyperactivity compared to wild-type controls, suggesting that genetic Cx36 deficiency may affect seizure susceptibility in a region-specific manner. Regions that showed increased PTZ sensitivity include the dorsal telencephalon, which is implicated in human epilepsy, and the lateral hypothalamus, which has been underexplored. We also found that PTZ-induced neuronal hyperactivity resulted in a rapid reduction of Cx36 protein levels. 30 minutes and one-hour exposure to 20 mM PTZ significantly reduced the expression of Cx36. This Cx36 reduction persists after one-hour of recovery but recovered after 3-6 hours. This acute downregulation of Cx36 by PTZ is likely maladaptive, as acute pharmacological blockade of Cx36 by mefloquine results in increased susceptibility to PTZ-induced neuronal hyperactivity. Together, these results demonstrate a reciprocal relationship between Cx36 and seizure-associated neuronal hyperactivity: Cx36 deficiency contributes region-specific susceptibility to neuronal hyperactivity, while neuronal hyperactivity-induced downregulation of Cx36 may increase the risk of future epileptic events.

neuroscience↗