The N-terminal 30–95^th amino acid region of RCAN1 is critical for HDAC3 binding.

<p>(<b>A</b>) Diagram of HA-tagged wild-type RCAN1 and its deletion mutants. RCAN1 consists of an N-terminal amphipathic leucine repeat (L) domain, a central span of 31 amino acids containing a serine-proline (SP) repeat, a C-terminal acidic region (a), and a cluster of basic amino acids (b). AS denotes the alternative splicing site of <i>RCAN1</i> between exon 1 or 4 and exon 5. (<b>B</b>) HEK293 cells were transfected for 24 hr with Flag-HDAC3 alone or in combination with various HA-tagged deletion RCAN1 mutants and treated for 6 hr with 10 µM MG132, as indicated. Total lysates and anti-Flag immunoprecipitates were analyzed by immunoblot using anti-HA or anti-Flag antibodies.</p

The stabilizing effect of HDAC3 is dependent on the RCAN1 N-terminal 30–95th amino acid.

<p>Where indicated, HEK293 cells were transfected for 24 hr with Flag-HDAC3 alone or together with HA-tagged wild-type RCAN1, RCAN11-95, RCAN11-125, RCAN130-197 or RCAN196-197 fragments. Cells were lysed in the lysis buffer containing 8 M urea, and immunoblot analyses were performed using HA and Flag antibodies. The HSP90 antibody was used as a loading control.</p

RCAN1 binds HDAC3 in HEK293 cells.

<p>(<b>A</b>) Where indicated, HEK293 cells were mock-transfected or transfected with plasmids encoding HA-tagged RCAN1 and/or Flag-tagged HDAC3 for 24 hr. Cell lysates were immunoprecipitated using anti-HA and anti-Flag antibodies, and immunocomplexes were analyzed by Western blotting using anti-HA or anti-Flag antibodies. Expression of transiently transfected proteins in cell lysates was identified using immunoblot analyses, as indicated. (<b>B, C</b>) HEK293 cell lysates were immunoprecipitated with anti-RCAN1 (<b>B</b>), anti-HDAC3 antibodies (<b>C</b>), or normal rabbit IgGs followed by immunoblotting using anti-HDAC3 and anti-RCAN1 antibodies, as indicated (*, nonspecific bands).</p

HDAC3 targets to and deacetylates RCAN1.

<p>Where indicated, HEK293 cells were transfected for 24 hr with HA-RCAN1 and/or Flag-HDAC3 (H3), and treated for 3 hr with 3 µM trichostatin A (TSA). Cell lysates were immunoprecipitated with anti-acetyl-Lys antibodies, followed by immunoblotting with anti-HA antiserum. Total lysates were analyzed by immunoblot using anti-HA or Flag antibodies. The HSP90 antibody was used as a loading control.</p

HDAC3 overexpression increases RCAN1 stability by inhibiting RCAN1 ubiquitination.

<p>(<b>A</b>) HEK293 cells were transfected for 24 hr with HA-tagged RCAN1 alone or in combination with increasing amounts of Flag-HDAC3. RCAN1 and HDAC3 levels were examined by immunoblotting with anti-HA and anti-Flag antibodies. The HSP90 antibody was used as a loading control. (<b>B</b>) HEK293 cells were mock-transfected or transfected with Flag-HDAC3 for 24 hr. Whole cell lysates were subjected to western blot analysis with anti-RCAN1, anti-Flag, and anti-HSP90 antibodies (*, nonspecific bands). (<b>C</b>) HEK293 cells were transfected for 24 hr with HA-RCAN1 and/or Flag-HDAC3 and cell extracts were lysed with Triton X-100 (soluble) and SDS (insoluble)-containing buffer. Each fraction was analyzed by immunoblotting with anti-HA or anti-Flag antibodies. To demonstrate equal loading, cell lysates were analyzed with anti-Hsp90 antibody, as indicated. (<b>D, E</b>) Where indicated, HEK293 cells were transfected for 24 hr with HA-RCAN1 or Flag-HDAC3 alone or in combination. Cells were treated with 100 µM cycloheximide (CHX) for the indicated times and harvested in PBS. RCAN1 levels in each sample were determined by western blot analyses using anti-HA antibodies (<b>D</b>). Data are representative of three independent experiments. Relative RCAN1 protein levels were quantified using the Multi Gauge V 3.1 program (*, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; <b>E</b>). (<b>F, G</b>) HEK293 cells were transfected for 24 hr with HA-RCAN1 or Flag-HDAC3 alone or in combination and treated for 6 hr with 10 µM MG132, as indicated. Cell lysates were immunoprecipitated using anti-HA antibody and immunoblotted using anti-ubiquitin or anti-HA antibodies. Proper expression of ubiquitin, RCAN1, or HDAC3 in cell extracts was determined by immunoblotting with their antibodies (<b>F</b>). Data are representative of three independent experiments. Relative ubiquitinated RCAN1 protein levels were quantified using the Multi Gauge V 3.1 program (*, <i>p</i><0.05; <b>G</b>).</p

The N-terminal 30–95^th amino acid region of RCAN1 is critical for HDAC3 binding.

<p>(<b>A</b>) Diagram of HA-tagged wild-type RCAN1 and its deletion mutants. RCAN1 consists of an N-terminal amphipathic leucine repeat (L) domain, a central span of 31 amino acids containing a serine-proline (SP) repeat, a C-terminal acidic region (a), and a cluster of basic amino acids (b). AS denotes the alternative splicing site of <i>RCAN1</i> between exon 1 or 4 and exon 5. (<b>B</b>) HEK293 cells were transfected for 24 hr with Flag-HDAC3 alone or in combination with various HA-tagged deletion RCAN1 mutants and treated for 6 hr with 10 µM MG132, as indicated. Total lysates and anti-Flag immunoprecipitates were analyzed by immunoblot using anti-HA or anti-Flag antibodies.</p

HDAC3 induces nuclear translocation of cytosolic RCAN1.

<p>(<b>A, B</b>) HEK293 cells were transfected for 24 hr with HA-RCAN1 and/or Flag-HDAC3 and fractionated into cytosolic and nuclear fractions. The fractions were analyzed by immunoblot using anti-HA or anti-Flag antibodies. The purity of each fraction was confirmed by immunoblotting with α-tubulin (cytosolic marker) or histone H1 (nuclear marker) (<b>A</b>). Data are representative of three independent experiments. Relative cytosolic RCAN1 and nuclear RCAN1 protein levels were quantified using the Multi Gauge V 3.1 program (**, <i>p</i><0.01; <b>B</b>). (<b>C</b>) HEK293 cells were transfected for 24 hr with HA-RCAN1 or/and Flag-HDAC3, fixed and permeabilized, and labeled with anti-HA or Flag antibodies. The cells were then stained with Alexa Fluor 488-conjugated anti-mouse and Alexa Fluor 594-conjugated anti-rabbit secondary antibodies. Nuclei were counterstained with DAPI, and immunostained preparations were visualized by confocal microscopy. Scale bars: 10 µm (<b>D</b>) HEK293 cell were transfected for 24 hr with HA-RCAN1 and/or Flag-HDAC3, and fractionated into the cytosolic and nuclear fractions. These fractions were immunoprecipitated with anti-acetyl-Lys antibodies, followed by immunoblotting with anti-HA antiserum. The expression of exogenously added RCAN1 or HDAC3 protein in each fraction was analyzed by immunoblotting anti-HA or Flag antibodies. The purity of each fraction was confirmed by immunoblotting with α-tubulin (cytosolic marker) or histone H3 (nuclear marker).</p

Autophagy is a novel pathway for neurofilament protein degradation <i>in vivo</i>

How macroautophagy/autophagy influences neurofilament (NF) proteins in neurons, a frequent target in neurodegenerative diseases and injury, is not known. NFs in axons have exceptionally long half-lives in vivo enabling formation of large stable supporting networks, but they can be rapidly degraded during Wallerian degeneration initiated by a limited calpain cleavage. Here, we identify autophagy as a previously unrecognized pathway for NF subunit protein degradation that modulates constitutive and inducible NF turnover in vivo. Levels of NEFL/NF-L, NEFM/NF-M, and NEFH/NF-H subunits rise substantially in neuroblastoma (N2a) cells after blocking autophagy either with the phosphatidylinositol 3-kinase (PtdIns3K) inhibitor 3-methyladenine (3-MA), by depleting ATG5 expression with shRNA, or by using both treatments. In contrast, activating autophagy with rapamycin significantly lowers NF levels in N2a cells. In the mouse brain, NF subunit levels increase in vivo after intracerebroventricular infusion of 3-MA. Furthermore, using tomographic confocal microscopy, immunoelectron microscopy, and biochemical fractionation, we demonstrate the presence of NF proteins intra-lumenally within autophagosomes (APs), autolysosomes (ALs), and lysosomes (LYs). Our findings establish a prominent role for autophagy in NF proteolysis. Autophagy may regulate axon cytoskeleton size and responses of the NF cytoskeleton to injury and disease.</p