FIGURE 10:

Phosphorylation of KalSR4:6 by Abl1 increases its sensitivity to calpain. (A) KalSR4:6 phosphorylated with [γ-³²P]ATP by Abl1 or exposed to the same conditions in the absence of ATP and Abl1 was exposed to the indicated amount of μ-calpain for 60 min at 37°C. Digested samples were fractionated by SDS–PAGE and transferred to polyvinylidene fluoride (PVDF) membranes; ³²P-labeled KalSR4:6 was visualized by autoradiography (left), and unlabeled KalSR4:6 was visualized by Western blot (right) using an antibody to SR4:7 (JH2580; Penzes et al., 2000). The amount of intact KalSR4:6 remaining was quantified; error bars show range of duplicate experiments. (B) Purified recombinant KalSR4:6 and KalSR4:6 with a phosphomimetic mutation at position 591 (SR4:6/Y591E) or 616 (SR4:6/Y616E) was fractionated by SDS–PAGE, transferred to a PVDF membrane, and visualized with Coomassie brilliant blue; mutation of either Tyr residue altered the mobility of the protein during electrophoresis. The purified proteins (1 μg each) were exposed to the indicated dose of μ-calpain as described. The reaction was stopped, and the SDS–PAGE fractionated products were visualized with Coomassie; mutation of either site increased the calpain sensitivity of the protein. (C, D) DIV18 cortical neurons from WT (C) and Kal7^KO (D) mice were exposed to medium containing 10 μM GNF-5 in DMSO, or an equivalent volume of DMSO, for 6 h and fixed. Images obtained using an Axiovert 200M with Apotome for optical sectioning were coded and scored by a blinded observer. GNF-5 increased the dendritic protrusion density in WT but not Kal7^KO neurons (C) while increasing the protrusion length in Kal7^KO but not WT neurons (D). N = 9–14 WT neurons and 6–8 Kal7^KO neurons; N = 1187–1652 WT protrusions and 306–838 Kal7^KO protrusions.