In Vitro and In Vivo Phosphorylation Assay For in vitro analysis of KIF3A phosphorylation by CAPK, HA-KIF3A was expressed in HEK293T cells, affinity-purified from cell components by anti-HA Sepharose beads. be intrinsically disordered. gene have been recognized in ciliopathies [14,15,16]. Our mouse model transporting such a ciliopathy mutation (R272Q) in the gene died at birth and displayed developmental abnormalities in multiple organ systems, demonstrating that is essential for embryonic development [17,18]. Given that the essential part of ICK is in the primary cilium and is associated with ciliopathy, we hereinafter refer to ICK as CAPK, ciliopathy-associated protein kinase. The molecular mechanisms underlying CAPK signaling and ciliary functions are still mainly unfamiliar. In main cilia, kinesin-2 engine complex (KIF3A/KIF3B/KAP3) mediates anterograde intraflagellar transport (IFT) which is critical for cilium formation and maintenance [19]. KIF3A has been proposed as a direct substrate of CAPK [7]. Here, we demonstrate that CAPK interacts with human being KIF3A and phosphorylates a conserved site Thr672 both in vitro and in vivo. RU 24969 We found that the long, unstructured, non-catalytic carboxyl-terminal website (CTD) of CAPK is required for this connection with and phosphorylation of KIF3A. We also provide persuasive evidence the CTD of CAPK is essential for not only its ciliary focusing on but also its part like a suppressor of ciliogenesis. 2. Materials and Methods 2.1. Plasmids and Antibodies pEBG-GST-CAPK plasmids encoding CAPK crazy type (WT), kinase lifeless (KD), and CTD truncation (1C291), as well as pEGFP-CAPK plasmids encoding CAPK WT, KD, R272A, and CTD truncation (1C291) were explained in [2,3]. pCIG-HA-KIF3A was explained in [20]. KIF3A-phospho-Thr672 antibody was generated in rabbits against keyhole limpet hemocyanin-coupled phospho-KIF3A peptide RPR[pT]SKGKARPKTGC at GenScript (Piscataway, RU 24969 NJ, USA). Phosphopeptide-specific antibodies were affinity-purified through a positive selection over phosphopeptide antigens followed by bad selections over non-phosphopeptide antigens. GST-tag (B-14) mouse monoclonal (sc-138) and HA-tag (12CA5) mouse monoclonal (sc-57592) antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). KIF3A (D7G3) rabbit monoclonal (#8507) and HA-tag (C29F4) rabbit monoclonal (#3724) antibodies were from Cell Signaling Technology (Danvers, MA, USA). Arl13B rabbit polyclonal antibody (17711-1-AP) was from Proteintech (Rosemont, IL, USA). Goat anti-rabbit IgG (Alexa Fluor 594) preadsorbed antibody (ab150084) was from Abcam (Cambridge, MA, USA). 2.2. Cell Tradition and Transfection HEK293T and NIH-3T3 cells were managed at 37 C and 5% CO2 in Dulbeccos altered Eagles RU 24969 medium (DMEM) supplemented with 4.5 g/L glucose and 10% fetal bovine serum (FBS) or 10% new given birth to calf serum (NBCS). HEK293T cells were transfected using a calcium phosphate protocol as explained in [21], and NIH-3T3 cells were transfected using the lipofectamine 2000 reagent following a manufacturers training. 2.3. GST Pull-Down, Immunoprecipitation, and Immunoblotting Forty eight hours after transfection, cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EGTA, complete protease inhibitors [Roche], 10 mM sodium orthovanadate, 5 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM -glycerophosphate, and 1 M microcystin LR). Cell lysate was cleared by centrifugation. GST-CAPK proteins were drawn down from cell lysate using glutathione Sepharose 4B beads (GE Healthcare, Chicago, IL, USA) following a manufacturers training. HA-KIF3A proteins were immunoprecipitated from cell lysate using the HA antibody, and captured on GammaBind Sepharose beads (GE Healthcare). Cell components or Sepharose beads were boiled for 5 min in an equal volume of 2X Laemmli sample buffer (120 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% -mercaptoethanol, 0.02% bromophenol blue) and loaded on an SDS gel. Samples were transferred to a PVDF (polyvinylidene difluoride) membrane and clogged for one hour in 5% dry milk before main antibody incubation in TBS RU 24969 comprising 0.1% Tween-20 and 5% bovine serum albumin (BSA) for 90 min at room temperature or overnight at 4 C. This was followed by considerable rinses and one hour incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. Chemiluminescence signals were developed using Millipore Immobilon ECL reagents (EMD Millipore, Burlington, MA, USA). 2.4. In Vitro and In RU 24969 Vivo Phosphorylation Rabbit polyclonal to Bcl6 Assay For in vitro analysis of KIF3A phosphorylation by CAPK, HA-KIF3A was indicated in HEK293T cells, affinity-purified from cell components by anti-HA Sepharose beads. HA-KIF3A substrates (0.1C0.5 g) were incubated with active His-CAPK1C291 [4,22] proteins (50 ng) and 100 M [-32P]-ATP (PerkinElmer) in kinase.
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