Regulation of synaptojanin 1 by cyclin-dependent kinase 5 at synapses

Abstract

Synaptojanin 1 is a polyphosphoinositide phosphatase concentrated in presynaptic nerve terminals, where it dephosphorylates a pool of phosphatidylinositol 4,5-bisphosphate implicated in synaptic vesicle recycling. Like other proteins with a role in endocytosis, synaptojanin 1 undergoes constitutive phosphorylation in resting synapses and stimulation-dependent dephosphorylation by calcineurin. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates synaptojanin 1 and regulates its function both in vitro and in intact synaptosomes. Cdk5 phosphorylation inhibited the inositol 5-phosphatase activity of synaptojanin 1, whereas dephosphorylation by calcineurin stimulated such activity. The activity of synaptojanin 1 was also stimulated by its interaction with endophilin 1, its major binding partner at the synapse. Notably, Cdk5 phosphorylated serine 1144, which is adjacent to the endophilin binding site. Mutation of serine 1144 to aspartic acid to mimic phosphorylation by Cdk5 inhibited the interaction of synaptojanin 1 with endophilin 1. These results suggest that Cdk5 and calcineurin may have an antagonistic role in the regulation of synaptojanin 1 recruitment and activity, and therefore in the regulation of phosphatidylinositol 4,5-bisphosphate turnover at synapses.

Fig. 1.

Purification of synaptojanin 1 from rat brain extract. (A) An immobilized GST fusion protein of the SH3 domain of endophilin 1 was used to affinity-purify binding proteins from a Triton X-100 rat brain extract. Bound proteins, represented primarily by synaptojanin 1 (SJ1) and dynamin 1 (Dyn1), were eluted by the PP-19 peptide (corresponding to the endophilin binding peptide of synaptojanin 1). Mono-Q chromatography of the PP-19 eluate with a linear gradient of NaCl resulted in the separation of the two proteins. Each fraction was examined by SDS/PAGE followed by protein staining. (B) Purified synaptojanin 1 has 5-phosphatase activity when tested against water-soluble phosphoinositides [diC₈-PI(4)P or diC₈-PI(4,5)P₂] by using a malachite green-based assay for the detection of free phosphate.

Fig. 3.

Phosphorylation of synaptojanin 1 by Cdk5 inhibits its 5-phosphatase activity. (A) Synaptojanin 1 was incubated with the p35/Cdk5 complex in phosphorylation conditions (see Fig. 2) in the presence or absence of the Cdk5 inhibitor roscovitine (20 μM). Phosphorylation mixtures and their controls were then added to assay tubes containing water-soluble PI(4,5)P₂ substrates [diC₈–PI(4,5)P₂ and NBD₆-PI(4,5)P₂] and further incubated for 10–15 min at 37°C. PI(4,5)P₂ cleavage was monitored by analysis of NBD₆ fluorescence after TLC chromatography. Note that only PI(4)P is generated. In separate experiments (data not shown), the 5-phosphatase activity of synaptojanin 1 was monitored by the release of free phosphate from diC₈-PI(4,5)P₂ using a malachite green-based assay (47). (B) Pooled results of 5-phosphatase activity assays involving the TLC and the malachite green-based assay. (C) Antisynaptojanin 1 immunoprecipitates from rat brain extracts were incubated in the presence or absence of the p35/Cdk5 complex in phosphorylation conditions, and the resulting mixtures were analyzed for 5-phosphatase activity as described for A. The Western blot (Upper) indicates that the same amount of synaptojanin 1 was present in the two samples. (D) A result of 5-phosphatase activity measured by malachite green-based assay.

Fig. 4.

Synaptojanin 1 is a substrate for calcineurin, and its dephosphorylation by calcineurin increases its 5-phosphatase activity. (A) Purified synaptojanin 1 (Upper) or dynamin 1 (Lower) were first phosphorylated by p35/Cdk5 in the presence of [γ-³²P]ATP, then further incubated with calcineurin, and finally analyzed by SDS/PAGE and autoradiography. (B) Immunoprecipitates generated from rat brain extracts with control IgG and anti-synaptojanin 1 antibody were incubated with calcineurin and then analyzed for 5-phosphatase activity by TLC as described in the legend of Fig. 3A. Anti-synaptojanin 1 Western blotting (Upper) indicates that the similar amount of synaptojanin 1 was present in the “plus” and “minus” calcineurin assay conditions. (C) Pooled results of 5-phosphatase activity assays as described in the legend of Fig. 3B.

Fig. 2.

In vitro phosphorylation of synaptojanin 1 by Cdk5. Purified synaptojanin 1 was incubated with the p35/Cdk5 complex and [γ-³²P]ATP in phosphorylation assay buffer, and samples were analyzed by SDS/PAGE and autoradiography. (A) Autoradiogram showing phosphorylation of synaptojanin 1 (SJ1) and p35 by Cdk5 after 60-min incubation. (B) γ-³²P autoradiogram showing a time-course of synaptojanin 1 and p35 phosphorylation.

Fig. 5.

Regulation of the 5-phosphatase activity of synaptojanin 1 in synaptosomes. (A) Synaptosomes were stimulated for 1 min with high K⁺ in the absence or presence of 2 μM cyclosporin A. Aliquots of synaptosomes stimulated without cyclosporin A were returned for 10 min to control medium or to control medium containing butyrolactone I (10 μM) or Ro31–8220 (20 μM). After solubilization of synaptosomes and anti-synaptojanin 1 immunoprecipitation, the 5-phosphatase activity of the immunoprecipitates was assayed with the malachite green-based assay. Bar graphs show average and range of two experiments. (B) A mixture of purified synaptojanin and dynamin was phosphorylated by p35/Cdk5 in the presence of [³²P]ATP and in the absence and presence of butyrolactone I or Ro31–8220 (20 μM each) and then analyzed by SDS/PAGE and autoradiography. Note that Ro31–8220 is more potent than butyrolactone I in inhibiting Cdk5 activity.

Fig. 6.

Cdk5 phosphorylation of the serine 1144 of rat synaptojanin 1 affects its interaction with endophilin and amphiphysin. (A) Serine 1144 and/or threonine 1147 were mutated to alanine, and GST fusions of the wild-type and mutant PRDs were incubated with p35/Cdk5 and [³²P]ATP. Incubation mixtures were then analyzed by SDS/PAGE followed by Coomassie blue (CB) staining and autoradiography. (B) GST fusion proteins of the wild-type PRD of synaptojanin 1, or of two mutants harboring a S → A or S → D mutation at position 1144, were used in pull-down assays from rat brain cytosol. Western blotting of the bound material revealed that the “phosphomimetic mutation” (S1144D) inhibited the recovery of both amphiphysin 1 and endophilin 1 in the bound material.

Fig. 7.

Stimulatory effect of endophilin 1 on the 5-phosphatase of synaptojanin 1. Purified synaptojanin 1 (0.2 μg) was preincubated for 30 min at 37°C with GST alone, GST-EndoSH3, or GST-endophilin 1 (GST-Endo1) at a molar concentration of 1:10 (synaptojanin 1/GST fusion proteins). The 5-phosphatase of this material was then assayed by either the TLC assay (A) or the malachite green-based assay (see Fig. 3A). The bar graphs shown in B represent the combined results from the two assays.