Protein kinase Cepsilon actin-binding site is important for neurite outgrowth during neuronal differentiation

Abstract

We have previously shown that protein kinase Cepsilon (PKCepsilon) induces neurite outgrowth via its regulatory domain and independently of its kinase activity. This study aimed at identifying mechanisms regulating PKCepsilon-mediated neurite induction. We show an increased association of PKCepsilon to the cytoskeleton during neuronal differentiation. Furthermore, neurite induction by overexpression of full-length PKCepsilon is suppressed if serum is removed from the cultures or if an actin-binding site is deleted from the protein. A peptide corresponding to the PKCepsilon actin-binding site suppresses neurite outgrowth during neuronal differentiation and outgrowth elicited by PKCepsilon overexpression. Neither serum removal, deletion of the actin-binding site, nor introduction of the peptide affects neurite induction by the isolated regulatory domain. Membrane targeting by myristoylation renders full-length PKCepsilon independent of both serum and the actin-binding site, and PKCepsilon colocalized with F-actin at the cortical cytoskeleton during neurite outgrowth. These results demonstrate that the actin-binding site is of importance for signals acting on PKCepsilon in a pathway leading to neurite outgrowth. Localization of PKCepsilon to the plasma membrane and/or the cortical cytoskeleton is conceivably important for its effect on neurite outgrowth.

Figure 1

Induction of neurite outgrowth by overexpression of PKCε. (A) SK-N-BE(2) neuroblastoma cells were transfected with expression vectors encoding full-length (FL) and RD from PKCδ, ε, η, and θ, fused to EGFP. Empty EGFP vector (−) was used as control. Cells were fixed 16 h after transfection and transfected cells with neurites longer than the length of two cell bodies were counted. Data (mean ± SEM, n = 2) are presented as percentage of transfected cells with long neurites. (B) Amount of EGFP fusion proteins in individual cells was analyzed with laser scanning cytometry. For each experiment, 50–150 cells were analyzed and the average of these cells was used as the observation value from that experiment. Data (mean ± SEM) are presented as arbitrary fluorescence intensity units from two separate experiments. (C) Cells expressing full-length PKCε or the PKCε RD fused to EGFP with fluorescence intensity of 350,000–550,000 U were scored for the presence of neurites. Twenty cells were scored in each experiment and data are presented as percentage of cells with neurites (mean ± SEM, five separate experiments) and arbitrary fluorescence intensity units (mean ± SEM, 100 different cells). SK-N-BE(2) cells were grown with or without 10% serum (D) or in serum-free medium with or without 16 nM TPA (E) for 16 h after transfection with vectors encoding EGFP (−), full-length PKCε (FL), or the PKCε RD (RD). Transfected cells were scored for neurites longer than two cell bodies and data, mean ± SEM, n = 3 (D) or 5 (E), are presented as percentage of transfected cells with neurites.

Figure 2

Association of PKCε with the cytoskeleton during neuronal differentiation. SK-N-BE(2) cells were induced to differentiate with 10 μM RA for 4 d. (A) Total cell lysates, normalized for total protein content, were analyzed for PKCε immunoreactivity and band intensities were quantified. Data (mean ± SEM, n = 4) represent PKCε levels in differentiated cells in percentage of values obtained in control cell lysates. (B) Lysed cells were divided into a Triton X-100 soluble cytosolic/membrane and a particulate cytoskeletal fraction. Cytoskeletal fractions, normalized to the Triton X-100-soluble fractions for protein content, were subjected to Western blot analysis by using anti-PKCε (top) and anti-actin (bottom) antibodies. Data (mean ± SEM, n = 10) are PKCε/actin intensity ratios from differentiated cells in percentage of corresponding values obtained from control cells. The increase is statistically significant (p < 0.05) using Student's t test.

Figure 3

Actin-binding site is of importance for neurite induction by full-length PKCε. (A) PKCε was modified by removing nucleotides encoding amino acids 223–228, i.e., the ABS, from wild-type PKCε cDNA (wt) and replacing it with an MluI recognition sequence, coding for amino acids T and R. This mutated PKCε (ΔABS) was also used to generate the new construct εRDΔABS. Neurite outgrowth was examined in SK-N-BE(2) (B) and SH-SY5Y cells (C) transfected with vectors encoding wild-type εFL and εRD (wt) and corresponding proteins with deleted actin-binding site (ΔABS), all fused to EGFP. The cells were fixed and mounted 16 h after transfection and transfected cells with long neurites were counted. Data (mean ± SEM, n = 3–6) are presented as percentage transfected cells with long neurites. ∗, statistically significant differences with analysis of variance followed by Duncan's multiple range test.

Figure 4

Isolated actin-binding site from PKCε inhibits neurite outgrowth. (A) Expression vectors encoding the actin-binding site from PKCε, or a scrambled version of this site, fused to either EGFP or a myc-tag via a linker sequence was constructed. (B) SK-N-BE(2) cells were cotransfected in a 1:7 ratio with vectors encoding EGFP, εFL-EGFP, or εRD-EGFP and myc-tagged ABS (ABS), scrambled ABS (Scram), or empty myc-vector (−). Cells were fixed 16 h after transfection and transfected cells, identified with EGFP fluorescence, were counted and the number of cells with long neurites was determined. Data (mean ± SEM, n = 3) are presented as percentage of transfected cells with long neurites. EGFP-tagged ABS, scrambled actin-binding site (Scram), and EGFP alone (EGFP) were expressed in SK-N-BE(2) cells (C) and SH-SY5Y/TrkA cells (D). The cells were incubated in regular medium (−) or treated with 10 μM RA for 2 d or with 100 ng/ml NGF for 4 d (RA, NGF). The cells were fixed and the number of transfected cells bearing long neurites was assessed. Data (mean ± SEM, n = 3) are presented as percentage of transfected cells with long neurites. ∗, statistically significant differences with analysis of variance followed by Duncan's multiple range test compared with similar conditions by using control vector instead of ABS vector.

Figure 5

Pseudosubstrate mutation does not compensate for deletion of the actin-binding site. (A) Nucleotide sequence encoding the pseudosubstrate sequence of PKCε with and without actin-binding site was mutated so that alanine 159 was changed to glutamate. (B) SK-N-BE(2) cells were transfected with vectors encoding EGFP fusions of PKCε, PKCε E159, PKCεΔABS, and PKCεΔABS E159, and the number of cells with neurites longer than two cell bodies was counted. Data (mean ± SEM, n = 4) are presented as percentage of transfected cells with neurites longer than two cell bodies.

Figure 6

Colocalization of PKCε and F-actin in growth cones. SK-N-BE(2) cells were transfected with vector encoding PKCε fused to EGFP (A and B) or treated with 10 μM RA for 3 d (C and D). Cells were fixed and F-actin was visualized with Alexa Fluor 546-conjugated phalloidin (B and D) and PKCε was visualized using EGFP fluorescence (A) or by immunofluorescence with Alexa Fluor 488-conjugated antibodies (C).

Figure 7

Negative effects of deletion of the actin-binding site on neurite induction are reversed by myristoylation of PKCε. (A) Localization to the plasma membrane of the isolated RD from PKCε fused to EGFP expressed in SK-N-BE(2) cells was shown with confocal microscopy. (B) cDNA encoding a myristoylation sequence derived from Lyn was fused to cDNA encoding εFL and εFLΔABS. In the schematic representation of the PKCε-EGFP vector, the BglII site used for cloning and the PKCε Kozak sequence precede the first codon, labeled 1. In the myristoylated PKCε (myrεFL) a PKCε Kozak sequence and a sequence encoding the first 10 aa from Lyn (boxed) are inserted into the NheI and BglII sites. The original Kozak sequence and BglII site are now translated. The original starting methionine is labeled 1. (C) SK-N-BE(2) cells were transfected with expression vectors encoding εFL, εFLΔABS, and the corresponding myristoylated variants (myr), all fused to EGFP. The cells were grown for 16 h and thereafter fixed, mounted, and examined with confocal microscopy. (D) Percentage of transfected cells with long neurites was quantified in cells expressing EGFP alone, PKCε RD (εRD), full-length PKCε (εFL), myristoylated PKCε (myrεFL), PKCε without the actin-binding site (εFLΔABS), and myristoylated PKCε without the actin-binding site (myrεFLΔABS). After transfection, the cells were cultured for 16 h with or without 10% serum. Data (mean ± SEM, n = 5–6) are presented as percentage of transfected cells with long neurites. (E) Expression levels in single cells of the EGFP fusion proteins in D were quantified with laser scanning cytometry. Data (mean ± SEM, n = 3, 50–100 cells analyzed in each experiment) are arbitrary units of fluorescence intensity. ∗, statistically significant differences with analysis of variance followed by Duncan's multiple range test.

Figure 8

Effects on the subcellular localization of PKCε by deletion of the actin-binding site. SK-N-BE(2) cells transfected with vector encoding full-length PKCε (A and C–E) and PKCε without the actin-binding site (B and F–H), both fused to EGFP. Transfected cells were subjected to subcellular fractionation (A and B) and divided into a cytosolic fraction (C), a membrane fraction (M), and a cytoskeletal fraction (S), which were analyzed with Western blotting with antibodies directed toward PKCε. Immunoblots from three individual experiments were quantified and data are presented as percentage of PKCε in each fraction out of total PKCε content. White bars represent endogenous PKCε and black bars represent EGFP-tagged PKCε variants. Cells expressing wild-type PKCε (C–E) and PKCε without actin-binding site (F-H) were fixed, and F-actin was stained with Alexa Fluor 546-conjugated phalloidin and by using confocal microscopy, a colocalization analysis was done. The images depict PKCε-EGFP (C and F), F-actin (D and G), and pixels that represent colocalization of PKCε and F-actin (E and H). Arrows highlight areas with cortical F-actin. The amount of phalloidin-positive pixels in the cortical cytoskeleton that was also EGFP-positive was 24% (wild-type PKCε) and 14% (PKCε with deleted actin-binding site) by using LaserPix software.

Figure 9

Disruption of F-actin leads to altered PKCε localization. SK-N-BE(2) cells were treated with 0.6 μM latrunculin B for 20 min whereafter PKCε was visualized with immunofluorescence (A and C) with Alexa Fluor 488-conjugated secondary antibodies, and F-actin was detected with Alexa Fluor 546-conjugated phalloidin (B and D). Cells were analyzed with a fluorescence microscope and images show control cells (A and B) and latrunculin B-treated cells (C and D). Arrows in A and B indicate cortical areas enriched in PKCε. These were invariably found in cortical areas where the cell had contact with another cell.