Reversible phosphorylation of Drp1 by cyclic AMP-dependent protein kinase and calcineurin regulates mitochondrial fission and cell death

Abstract

Opposing mitochondrial fission and fusion reactions determine the shape and interconnectivity of mitochondria. Dynamin-related protein 1 (Drp1) is an ancient mechanoenzyme that uses GTP hydrolysis to power the constriction and division of mitochondria. Although Drp1-mediated mitochondrial fragmentation is recognized as an early event in the apoptotic programme, acute regulation of Drp1 activity is poorly understood. Here, we identify a crucial phosphorylation site that is conserved in all metazoan Drp1 orthologues. Ser 656 is phosphorylated by cyclic AMP-dependent protein kinase and dephosphorylated by calcineurin, and its phosphorylation state is controlled by sympathetic tone, calcium levels and cell viability. Pseudophosphorylation of Drp1 by mutation of Ser 656 to aspartic acid leads to the elongation of mitochondria and confers resistance to various pro-apoptotic insults. Conversely, the constitutively dephosphorylated Ser656Ala mutant Drp1 promotes mitochondrial fragmentation and increases cell vulnerability. Thus, Drp1 phosphorylation at Ser 656 provides a mechanism for the integration of cAMP and calcium signals in the control of mitochondrial shape, apoptosis and other aspects of mitochondrial function.

Figure 1

Identification of Drp1 Ser 656 as a PKA phosphorylation site. (A) Endogenous Drp1 was immunoprecipitated (IP) from PC12 cells that had been metabolically labelled with ³²PO₄²⁻. Cells were treated with okadaic acid (OA, 300 nM, 2 h), calyculin A (Calyc A, 25 nM, 1 h), forskolin (20 μM, 1 h) or vehicle (control) before collection. The bar graph shows ³²P incorporation normalized to Drp1 levels and relative to control (mean±s.e.m. of n=3–8 experiments, ^*P<0.0005 by Student's t-test). (B) Domain diagram of Drp1 and sequence alignment of the boundary between the variable domain (VD) and the GTPase effector domain (GED; PKA consensus underlined). (C) GST–Drp1_GED (aa 643–755) fusion proteins were phosphorylated in vitro with PKA and [γ-³²P]ATP. (D) GFP and 3 × haemagglutinin (HA)-tagged wild-type (WT) and Ser656Ala Drp1 were expressed in COS cells metabolically labelled with ³²P and then immunoprecipitated with a Drp1 antibody. (E) 3 × HA–Drp1 (WT/Ser656Ala) was coexpressed with empty vector (−) or PKA_c (catalytic subunit) in COS cells metabolically labelled with ³²P and analysed after HA IP. Drp1, dynamin-related protein 1; GFP, green fluorescent protein; GST, glutathione S-transferase; GTP, GTPase domain; IB, immunoblotting; MID, middle domain; PKA, cAMP-dependent protein kinase.

Figure 2

Effects of phosphorylation site mutant Drp1 on mitochondrial morphology. (A) Epifluorescence micrographs of immunofluorescently labelled mitochondria from CV1 fibroblasts in which endogenous Drp1 was replaced with wild-type (WT), Ser656Ala and Ser656Asp GFP–Drp1 (transfected cells are outlined). (B) Quantification of the mitochondrial area in CV1 cells substituted with WT, Ser656Ala (A) and Ser656Asp (D) GFP–Drp1 by computer-aided image analysis (mean±s.e.m. of six experiments with 60–80 cells per condition each, normalized to WT). ^*P<0.01, ^**P<5 × 10⁻⁴ by Student's t-test. Drp1, dynamin-related protein 1; GFP, green fluorescent protein.

Figure 3

Regulation of Drp1 Ser 656 phosphorylation in cells and in vivo. (A) Mice received intraperitoneal injections of vehicle or isoproterenol (20 mg/kg; left panel) or were subjected to 15 min of forced swimming or rest (right panel). Drp1 was immunoprecipitated from rapidly dissected heart muscle and analysed for Ser 656 phosphorylation (pS656 Drp1) with a phosphospecific antibody. Multiple bands most likely correspond to splice variants. (B) PC12 cells were preincubated for 15 min with the phosphatase inhibitors FK506 (1 μM), cyclosporin A (CsA, 1 μM) or calyculin A (CaA, 20 nM) and then treated for 60 min with forskolin (10 μM) to activate PKA and 25 mM K⁺/0.1 μM BayK8644 (25K/BayK) to activate L-type Ca²⁺ channels. Total lysates were probed for phospho-Ser 656 and total Drp1. (C) PC12 cells preincubated for 15 min with FK506 (FK, 1 μM) or vehicle were treated for 60 min with forskolin (10 μM)±staurosporine (STS, 2 μM), followed by immunoblotting of total lysates as indicated. (D) PC12 cells stably expressing GFP–Drp1 WT or Ser656Ala were incubated with staurosporine (1 μM) for the indicated times (hours), and total lysates were analysed for Drp1 Ser 656 phosphorylation. Data are representative of at least three independent experiments. Drp1, dynamin-related protein 1; GFP, green fluorescent protein; PKA, cAMP-dependent protein kinase, pS40 TH, phospho-Ser 40 tyrosine hydroxylase.

Figure 4

Drp1 Ser 656 determines apoptotic sensitivity. PC12 cells stably substituting wild-type (WT) and Ser 656 mutant GFP–Drp1 for endogenous Drp1 were challenged for the indicated times (A) or for 48 h with the indicated doses of staurosporine or etoposide, followed by caspase activity (B) and viability (A,C,D) assays. Data (mean±s.d. of quadruplicate determinations) are representative of three or more independent experiments. Two to three clonal lines of each Drp1 genotype were analysed (two Drp1 Ser656Ala lines are shown here for comparison). Drp1, dynamin-related protein 1; GFP, green fluorescent protein.