Identification of a Ste20-like kinase-dependent signaling network orchestrating cytoskeletal and synaptic development of cortical neurons

Abstract

The Ste20-like kinase (SLK) is a serine/threonine kinase that regulates cytoskeletal organization, apoptosis, and cell polarity in non-neuronal cells. In neurons, SLK guides dendrite morphogenesis and the stability of inhibitory synapses, thereby controlling excitation-inhibition balance through parvalbumin interneuron function. Almost complete loss of SLK in dysmorphic neurons of epileptogenic cortical dysplasias implicates a function of this kinase in neurodevelopmental disorders. However, its neuronal signaling mechanisms remain poorly understood. To address this knowledge gap, we combined an affinity purification-mass spectrometry screen for candidate SLK-interacting proteins with quantitative phosphoproteomics after SLK knockdown to identify potential SLK interactors and the SLK-dependent phosphorylation landscape in cortical neurons. Across the two datasets, we identified hundreds of candidate SLK-binding proteins and more than 1,000 regulated phosphosites after SLK knockdown. The datasets converged on cytoskeletal, synaptic, and signaling proteins involved in actin and microtubule regulation, cell adhesion, and synaptic stability. We identified two germinal center (GCK)-family kinases, MINK1 and TNIK, as direct SLK substrates at the activation-loop threonine (T181), suggesting that SLK may act upstream of a GCK kinase pathway implicated in dendritic development. Functionally, SLK knockdown also altered spontaneous network activity, changed the phosphorylation pattern of KCNQ2, and increased the sensitivity of neuronal networks to KCNQ2 blockade. These data position SLK within a broad neuronal signaling network that links cytoskeletal and synaptic regulation to kinase signaling and neuronal excitability. While the effects on KCNQ2 appear to be indirect, MINK1 and TNIK emerge as direct SLK substrates.