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 multisite 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.

Keywords: RhoGEF; cocaine; mass spectrometry; nucleus accumbens; prefrontal cortex; tyrosine phosphorylation.

Figure 1. Kalirin isoforms and sample preparation

A. Kal7 consists of a Sec14 domain, 9 spectrin repeats (SR1 to SR9), a DH domain, a PH domain, a short, unstructured linker region and a PDZ binding motif. Biophysical studies predict a compact structure for Kal7^, . The structures of the other major isoforms are indicated; Kin, kinase. B. Adult male rats (n = 6-7/group) were subjected to saline, acute or chronic cocaine treatment. Animals were sacrificed 30 min after the final injection. Immunoprecipitates prepared from individual PFC and NAc samples (S1, A7, C14, etc.) were separated by SDS PAGE. C. Protein bands were visualized by silver staining; Kal7, Kal9 and Kal12 are indicated. Gel fragments containing Kal7 and a pool of Kal9 and Kal12 were excised and processed.

Figure 2. Identification of 26 phosphorylation sites in Kal7

A. Bar graph showing degree of phosphorylation of each site relative to the total amount of peptide recovered. Blue and green bars show phosphorylation of Ser/Thr residues in NAc and PFC, respectively; red and pink bars indicate the same for Tyr residues. Residues are indicated on the X axis, where ampersands (&) indicate indefinite assignment. The grey dashed line indicates 50% phosphorylation. Sites that differ significantly in extent of phosphorylation in NAc vs. PFC are indicated by a # (p <0.05) or * (p <0.002) above the bar. A schematic of Kal7 indicating the location of each phosphorylation site is shown below the bar graph. Sites are numbered using AF230644_1 for rat cKal7. B. Schematic identifies residues where phosphorylation was significantly different between NAc and PFC in saline injected control animals.

Figure 3. Identification of 30 phosphorylation sites in Kal9/12 from rat PFC

A. Schematic showing phosphorylation sites identified in Kal9/Kal12 from Saline-injected control rat PFC. Residue numbers are shown only for pY sites. Sites shared by Kal9/Kal12 are bracketed, as are sites unique to Kal12. B. Isoform-specific phosphorylation sites are depicted. Phosphorylation sites identified in Kal7 but absent from Kal9/Kal12 are indicated above the schematic. Phosphorylation sites identified in Kal9/Kal12 but absent from Kal7 are shown below the schematic.

Figure 4. Screening pY peptides with multiple GST-SH2 constructs

A. Seventy-four phosphotyrosine interacting domains (SH2, PTP, and PTB) and a GST control were tested. The probe list is provided in Supplemental Table S2. The sequences of the four internal Kal7 pY peptides tested are shown. B. Overview of SH2 screen. Four pairs of phosphorylated and non-phosphorylated peptides were immobilized in a rosette pattern; after incubation with the GST-tagged probes, signal was detected with infrared (IR) dye labeled secondary antibodies. C. Representative IR scan data are shown; green signal indicates probe binding and red signal monitors peptide loading using streptavidin. The pY antibody control confirms the phosphorylation state of the biotinylated peptide. The GST control assesses background. In this panel, Fyn-SH2 bound strongly to pY1123 and pY1342, while Csk-SH2 bound only to pY616. D. Quantified results for pY, Fyn-SH2, Csk-SH2, and GST probes. Signal for each peptide was normalized to the streptavidin signal for that peptide; values plotted are the average of quadruplicates with SEM. As seen in the streptavidin signal, the pY1123 peptide bound relatively poorly to the membrane; the ratiometric quantification corrects for this.

Figure 5. Comparison of GST-SH2 binding data for different pY sites

A. Normalized SH2 binding to the four internal Kal7 pY sites are presented as a heatmap (positive binding in yellow); probes appear in reverse alphabetical order, left to right. Overall the pY peptides showed enhanced signal compared to their non-phosphorylated Y peptide counterparts, indicating phosphorylation-dependent binding of the probes. Notably, pY1123 was recognized by more probes than the other peptides. Red asterisks indicate the pTyr and GST controls. B. Hierarchical clustering was used to identify probes exhibiting similar binding patterns; two subclusters were identified (SFK: Src family kinases; PTP: protein tyrosine phosphatases). C. Interactors for each pY peptide were ordered by signal intensity (strong interactors on the left) and the position of each pY in Kal7 is shown. The cut-off for positive binders was defined as >3× the GST background.

Figure 6. Far-Western analysis of selected interactors

A. Coomassie gel of purified bKal7. B. Purified bKal7 was tyrosine phosphorylated using TrkB. Presence of purified bKal7 protein (∼190 KDa), TrkB protein (∼70 KDa), and their tyrosine phosphorylation state were assessed by Western blot analysis. C. Validation of SH2 screen. Capillary far-Western assay was performed to confirm direct interactions between the probe and purified, phosphorylated bKal7. Red asterisks in blot view indicate the presence of phospho-Kal7 (∼190 KDa) with TrkB treatment; blue asterisks indicate TrkB (∼70 KDa). The higher molecular weight bands detected by several probes are non-specific. D. Summary of SH2 screen and capillary far-Western results for 12 SH2/PTP domains and control probes. Positive interactions are indicated, with red color proportional to strength.

Figure 7. Calpain cleavage of Kal7

A. Lysates (30 μl; ∼60 μg protein) of HEK cells expressing Kal7 were analyzed directly (Input, In) or digested with the indicated dose of μ-calpain (0 to 120 ng) at 37C for 1 h before denaturation with SDS and Western blot analysis using an affinity-purified antibody that recognizes SR4:7; markers are shown to the left and observed masses to the right. B. In additional experiments, Input and μ-calpain treated samples prepared with the highest amount of μ-calpain were analyzed using antibodies to the C-terminus (product masses shown in red) and Sec14 domain (product masses shown in blue) of Kal7. C. Input and μ-calpain-digested samples were analyzed using the SR4:7 antibody and the C-term antibody; after normalizing the C-term/SR4:7 ratio for each Input sample to 1.0, the ratio calculated for the corresponding calpain-digested sample was significantly less than 1.0 (p < 0.001; t-test), suggesting that intact Kal7 and a C-terminally truncated μ-calpain product were not being resolved by this gel system. D. Vector encoding the MycHis-tagged GEF1-end protein shown in the schematic, which extends from the GEF1 domain to the C-terminus was transiently expressed and analyzed as described for Panels A and B; the red arrow indicates the location of a high probability calpain site. Proteins were visualized with Myc antibody (left) or C-term antibody (right), demonstrating calpain-catalyzed removal of the C-terminal epitope from the 41K product. E. For each μ-calpain digest, the total amount of signal obtained with the SR4:7 or C-term antibody was determined; the % of that signal accounted for by each major product was determined (n = 4; error bars are std dev). F. μ-Calpain cleavage sites in rat Kal7 were predicted using GPS-CCD the 5 top sites, with scores > 1.0 are shown in thick red lines; sites with scores of 0.8 to 1.0 are shown in black. Cleavage at the most C-terminal calpain site generates a product just 3 amino acid longer than the unique region of Kal7. G. HEK293 cells transiently transfected with vector encoding aKal7 were treated with the indicated dose of staurosporine for 4 h before extraction for μ-calpain digestion. Staurosporine treatment increased the amount of intact Kal7 and reduced product formation.

Figure 8. Effect of single site mutations on calpain cleavage of Kal7

A. Diagram indicating the sites targeted for mutagenesis and the major μ-calpain cleavage sites; mutation of the sites indicated in red affected stability and/or cleavage. B. Ratio of 191K band intensity (SR4:7 Ab) for phosphomimetic vs. nonphosphorylatable Kal7 mutants expressed in HEK cells (n = 3 to 7; One-Way ANOVA by ranks). C. Lysates prepared from HEK cells expressing Kal7/Y591E were digested using different amounts of μ calpain, as in Fig. 7A. D. Lysates containing Kal7/Y591E or Kal7/Y591F were digested with μ-calpain under identical conditions, revealing more extensive cleavage of the Y591E mutant. Quantitative analysis of μ-calpain products of Kal7/Y591E and Kal7/Y591F detected using the C-term antibody; the digestion patterns differed significantly (One-Way ANOVA on Ranks; p<0.05 for 191K, 109K and 45K). E. Analysis of Kal7/Y1123E and Kal7/Y1123F mutants. Although consistently expressed less well than Kal7/Y1123F, Kal7/Y1123E yielded similar digestion products. F. Analysis of Kal7 with T1519S1520 mutated to ED or to AA. Kal7/TS/ED was poorly expressed compared to Kal7/TS/AA, and the stable products of μ-calpain digestion were barely detected in digests of Kal7/TS/ED (p<0.01, both antibodies, One-Way ANOVA by ranks).

Figure 9. Effect of phosphorylation on GEF activity

A. Cell-based DoraRac biosensor FRET assay. GEF1: WT vs. non-phosphorylatable mutant (quad: T1303A, Y1342F, TS/AA). B. GEF1→ end. WT vs. Y1653E and Y1653F, plotted using densitized protein values from Western analyses; inactive Kal-GEF/ND/AA gave no activity above Biosensor alone. C. Kal7 mutants: Y1653F and Quad (non-phosphorylatable) mutant, plotted using densitized protein values from Western analyses. D-F. Biosensor assays demonstrated that Kal7/Y591F and /Y591E were equally active in 3 assays. GEF assays demonstrated that Kal7 and Kal7/Y1123F were equally active in 5 assays in duplicate (p=0.85), while Kal7/Y1123E was significantly less active in 4 assays in duplicate (p <0.001). Kal7/TS/AA was fully active in 3 assays, but the TS/ED version of Kal7 was 3-20 times less active in 3 assays.

Figure 10. Cocaine-mediated changes in Kal7 phosphorylation in NAc and PFC

A. Diagram showing location of cocaine regulated phosphorylation sites in Kal7 isolated from NAc (above schematic) and PFC (below schematic). B. Table summarizing significant changes in degree of phosphorylation at cocaine-regulated sites in NAc and PFC Kal7.