Phosphorylation-dependent Regulation of Connecdenn/DENND1 Guanine Nucleotide Exchange Factors

Abstract

Connecdenn 1/2 are DENN (differentially expressed in normal and neoplastic cells) domain-bearing proteins that function as GEFs (guanine nucleotide exchange factors) for the small GTPase Rab35. Disruption of connecdenn/Rab35 function leads to defects in the recycling of multiple cargo proteins from endosomes with altered cell function, yet the regulation of connecdenn GEF activity is unexplored. We now demonstrate that connecdenn 1/2 are autoinhibited such that the purified, full-length proteins have significantly less Rab35 binding and GEF activity than the isolated DENN domain. Both proteins are phosphorylated with prominent phosphorylation sites between residues 500 and 600 of connecdenn 1. A large scale proteomics screen revealed that connecdenn 1 is phosphorylated at residues Ser-536 and Ser-538 in an Akt-dependent manner in response to insulin stimulation of adipocytes. Interestingly, we find that an Akt inhibitor reduces connecdenn 1 interaction with Rab35 after insulin treatment of adipocytes. Remarkably, a peptide flanking Ser-536/Ser-538 binds the DENN domain of connecdenn 1, whereas a phosphomimetic peptide does not. Moreover, connecdenn 1 interacts with 14-3-3 proteins, and this interaction is also disrupted by Akt inhibition and by mutation of Ser-536/Ser-538. We propose that Akt phosphorylation of connecdenn 1 downstream of insulin activation regulates connecdenn 1 function through an intramolecular interaction.

Keywords: DENN domain; Rab; Rab35; cell biology; connecdenn; endocytosis; endosome; guanine nucleotide exchange factor (GEF); intracellular trafficking; membrane trafficking.

FIGURE 1.

Isolated DENN domains of connecdenn 1 and 2 have greater GEF activity than the full-length proteins. A, FLAG-tagged full-length (FL) connecdenn 1 (CD 1) or its isolated DENN domain was immunoprecipitated from HEK-293 cell lysates along with immunoprecipitation (IP) from mock-transfected cells as a control. Aliquots of the immunoprecipitated proteins were processed for FLAG Western blot (right panel) or were added to the exchange reaction that contained [³⁵S]GTPγS and purified Rab35 preloaded with GDP (left panel). The enzymatic activity based on pmol of Rab35 loaded with GTPγS over time is shown. B, as for A except with connecdenn 2 (CD 2). C, GST or GST-Rab35 S22N (constitutively GDP-bound mutant) were incubated with HEK-293 lysates expressing FLAG-tagged full-length connecdenn 1 or its isolated DENN domain. Specifically bound proteins were processed for Western blot with FLAG antibody. Starting material (SM) represents 10% of input. D, as for C except with connecdenn 2.

FIGURE 2.

Connecdenn proteins are phosphorylated on serine and threonine residues. A, HEK-293 cells expressing FLAG-tagged full-length connecdenn 1 (FLAG-CD 1 FL) were treated with DMSO (vehicle control) or with the indicated concentrations of OA for 2 h and then processed for Western blot with FLAG antibody. B, HEK-293 cells expressing FLAG-connecdenn 1 full-length were treated with DMSO or with 250 nm OA and 500 nm Na₃VO₄ or with the indicated concentrations of Na₃VO₄ alone for 2 h and then processed for Western blot with FLAG antibody. C, as in A except with full-length FLAG-tagged connecdenn 2 (FLAG-CD 2 FL). D, HEK-293 cells expressing FLAG-connecdenn 1 full-length were preincubated in phosphate-free DMEM media for 2 h with 0.125 μCi of label-free [³²]P_i/15-cm plate and subsequently treated with 250 nm OA or DMSO for 2 h. FLAG-connecdenn 1 full-length was immunoprecipitated, resolved by SDS-PAGE, and subjected to autoradiography along with aliquots of the starting material (SM) equal to 5% or 0.5% of that added to the immunoprecipitation (IP). E, as in D but for FLAG-connecdenn 2 full-length.

FIGURE 3.

Connecdenn 1 is phosphorylated in the C terminus and phosphorylation promotes 14-3-3 binding. A, FLAG-tagged full-length (FL) connecdenn 1, deletion constructs with the indicated boundaries, the isolated DENN domain (residues 1–406), or the isolated C-terminal (CT) region (residues 375–1016) were expressed in HEK-293 cells. Cells were treated with either 250 nm OA or DMSO (vehicle control) for 2 h. Total cell lysates were processed for Western blot with FLAG antibody. B, GST, GST-14-3-3 wild-type (WT) of GST-14-3-3 K49E mutant were incubated with HEK-293 lysates expressing FLAG-tagged full-length connecdenn 1 (FLAG-CD 1 FL). Specifically bound proteins were processed for Western blot (WB) with FLAG antibody. Starting material (SM) equals 10% of the input. C, GST or GST-14-3-3 wild-type were incubated with HEK-293 lysates expressing FLAG tagged full-length connecdenn 1 or various C-terminal deletion constructs as described in A. Before lysis, cells were treated with either 250 nm OA or DMSO for 2 h. Specifically bound proteins were processed for Western blot with FLAG antibody. Starting material equals 10% of the input.

FIGURE 4.

Akt inhibition reduces 14-3-3 binding. A, highly differentiated 3T3-L1 adipocytes were stimulated with 100 nm insulin for 30 min in the presence or absence of 10 μm MK2206, an Akt inhibitor administered 30 min before insulin treatment. After treatment, cell lysates were prepared and incubated with GST or GST-14-3-3 pre-coupled to glutathione-Sepharose beads, and specifically bound proteins were detected with an antibody recognizing endogenous connecdenn 1. SM, starting material. B, cell lysates prepared as in A were processed for Western blots with antibodies against the indicated proteins. C, quantification of the binding of connecdenn 1 to GST-14-3-3 as in A. The bar represents S.E. Statistical analysis employed an unpaired t test. p < 0.05.

FIGURE 5.

Akt inhibition reduces Rab35 binding. A, highly differentiated 3T3-L1 adipocytes were stimulated with 100 nm insulin for 30 min in the presence or absence of 10 μm MK2206, an Akt inhibitor administered 30 min before insulin treatment. After treatment, cell lysates were prepared and incubated with GST or GST-Rab35 S22N (constitutively GDP-bound mutant) pre-coupled to glutathione-Sepharose beads, and specifically bound proteins were detected with an antibody recognizing endogenous connecdenn 1. SM, starting material. B, cell lysates prepared as in A were processed for Western blots with antibodies against the indicated proteins. C, quantification of the binding of connecdenn 1 to GST-Rab35 S22N as in A. The bar represents S.E. Statistical analysis employed an unpaired t test. *, p < 0.05.

FIGURE 6.

Ser-536 and Ser-538 contribute to interactions with the DENN domain and 14-3-3. A, FLAG-tagged connecdenn 1 DENN domain (FLAG-CD 1 DENN) was expressed in HEK-293 cells, and cell lysates were incubated with amylose resin coupled to MBP fused to lacZ or a connecdenn 1 peptide encoding residues 527–547, WT, or S536E/S538E mutant. Specifically bound proteins were processed for Western blot with FLAG antibody. Starting material (SM) equals 5% of the input. B, GST or GST-14-3-3 were incubated with HEK-293 lysates expressing FLAG tagged full-length connecdenn 1 (FLAG-CD 1) WT or S536E/S538E mutant. Specifically bound proteins were processed for Western blot with FLAG antibody. Starting material equals 10% of the input. C, quantification of the binding of connecdenn 1 to MBP-peptides as in B. The bar represents S.E. Statistical analysis was employed an unpaired t test. *, p < 0.05.