Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

Abstract

Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long term potentiation and in the effects of cocaine on behavior and spine morphology. The KALRN gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multi-site phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity and reduced GEF activity.

Dissecting the Roles of Kalirin-7/PSD-95/GluN2B Interactions in Different Forms of Synaptic Plasticity

“…1B,1C), mimicking the electrophysiological response observed in Kal7 knockout spinal neurons (Lu et al, 2015). It is intriguing that a very early cleavage of Kal7 by calpain produces a peptide virtually identical to one of the peptides tested (Kal7-L; Table 1) (Miller, Yan, Machida, et al, 2017). Intracellular calpain cleavages are established as critical for long-term potentiation (Briz & Baudry, 2016).…”

Dissecting the Roles of Kalirin-7/PSD95/GluN2B Interactions in Different Forms of Synaptic Plasticity

“…However, no competition could be demonstrated, despite various experimental approaches that included multiple resins and a wide range of concentrations of competing peptides. Covalent binding of PDZ123 to Affigel and to UltraLink Biosupport resins showed a dose-dependent saturable binding of Kal7 to the PDZ123 resin and little background binding to the BSA resin, using 5 pmol purified Kal7 input (Miller, Yan, Machida, et al, 2017) (Fig.7A, asterisks). However, competing GluN2B and Kal7 peptides had no effect on the binding of baculovirus Kal7 to the PDZ123 resin, even though binding of Kal7 to the BSA resin was minimal (Fig.7B).…”

“…Protein purity was verified by SDS-PAGE, followed by transfer to PVDF membranes, staining with Coomassie Brilliant Blue and Western blot analysis using a pan-MAGUK antibody (NeuroMab 75-028, clone 28/43; RRID AB_2292909 ) (Davis CA), a PSD-95 antibody (Upstate 05-427; RRID:AB_444362) (Lake Placid NY), and Kalirin antibodies JH2580 and JH2958 (Miller, Yan, Machida, et al, 2017) (RRID: 2801571;2801572). Western blots were quantified in the linear range of exposures as described (Miller, Yan, Machida, et al, 2017). Similar experiments were conducted using mammalian PSD-95 protein (prepared by transfecting expression vectors pCMV-PSD95-flag [FLAG-tagged; Addgene, #15463] into HEK293 cells) bound to GluN2B-peptide-containing Agarose resins, as well as the binding of purified Kal7 [from baculovirus expression (Miller, Yan, Machida, et al, 2017)] paired with competing GluN2B peptides and control peptides (several biotinylated peptides from within the Kal7 sequence).…”

“…Western blots were quantified in the linear range of exposures as described (Miller, Yan, Machida, et al, 2017). Similar experiments were conducted using mammalian PSD-95 protein (prepared by transfecting expression vectors pCMV-PSD95-flag [FLAG-tagged; Addgene, #15463] into HEK293 cells) bound to GluN2B-peptide-containing Agarose resins, as well as the binding of purified Kal7 [from baculovirus expression (Miller, Yan, Machida, et al, 2017)] paired with competing GluN2B peptides and control peptides (several biotinylated peptides from within the Kal7 sequence).…”

“…Resin was blocked with 1 mg/ml BSA for 15 min, washed in binding buffer, 0.6M Na-citrate (pH 7.4), binding buffer, then binding experiments were conducted and analyzed by Western blot (see below). For co-immunoprecipitation experiments, PSD95, Kal7, and Kal8 proteins were prepared by transfecting individual expression vectors into HEK293 cells: pCMV-PSD95-flag, an empty pFIV shuttle vector, pEAK-HisMyc-Kal7a, or pSCEP-Myc-Kal8 (Miller, Vishwanatha, Mains, & Eipper, 2015;Miller, Yan, Machida, et al, 2017). After 24 hours of transfection, HEK293 cells were rinsed in serum free medium (Rao, Zavala, Deb Roy, Mains, & Eipper, 2019), pelleted, and vortexed in ice-cold buffer containing 20mM TES, 10mM mannitol, 0.3mg/ml PMSF, 2μg/ml leupeptin, 2μg/ml pepstatin, 2 μg/ml benzamidine, 16μg/ml benzamidine, and 50μg/ml LBTI.…”