Neurabin is a synaptic protein linking p70 S6 kinase and the neuronal cytoskeleton

Abstract

p70 S6 kinase (p70(S6k)) is a mitogen-activated protein kinase that plays a central role in the control of mRNA translation. It physiologically phosphorylates the S6 protein of the 40s ribosomal subunit in response to mitogenic stimuli and is a downstream component of the rapamycin-sensitive pathway, which includes the 12-kDa FK506 binding protein and includes rapamycin and the 12-kDa FK506 binding protein target 1. Here, we report the identification of neurabin (neural tissue-specific F-actin binding protein), a neuronally enriched protein of 1,095 amino acids that contains a PDZ domain and binds p70(S6k). We demonstrate the neurabin-p70(S6k) interaction by yeast two-hybrid analysis and biochemical techniques. p70(S6k) and neurabin coimmunoprecipitate from transfected HEK293 cells. Site-directed mutagenesis of neurabin implicates its PDZ domain in the interaction with p70(S6k), and deletion of the carboxyl-terminal five amino acids of p70(S6k) abrogates the interaction. Cotransfection of neurabin in HEK293 cells activates p70(S6k) kinase activity. The mRNA of neurabin and p70(S6k) show striking colocalization in brain sections by in situ hybridization. Subcellular fractionation of rat brain demonstrates that neurabin and p70(S6k) both localize to the soluble fraction of synaptosomes. By way of its PDZ domain, the neuronal-specific neurabin may target p70(S6k) to nerve terminals.

Figure 1

Cloning and expression of neurabin cDNA. (a) Structure of neurabin and p70^S6k. The region of p70^S6k used to screen the two-hybrid library and the region of neurabin identified in the two-hybrid screen are indicated. Also shown are the two neurabin clones (λ ZAP #1 and #2) recovered from a rat brain library. These contain the entire ORF of the neurabin protein. (b) Deduced amino acid sequence from the neurabin cDNA. The PDZ domain is underlined, and the conserved amino acids Gly-Leu-Gly-Ile, which were mutated to Gly-Leu-Ala-Ala, are in bold print. (c) Expression of neurabin cDNA. Neurabin cDNA in pRK5 either was transcribed and translated in vitro or was transfected into HEK293 cells. Both generated protein that comigrates with the neurabin protein from brain lysate.

Figure 2

Neurabin interacts with p70^S6k both in vitro and in vivo. (a) In vitro binding of myc- p70^S6k from transfected HEK293 cells to a GST or GST-neurabin (amino acids 486–751) column. Salt concentrations of washes are indicated. (b) Coimmunoprecipitation of myc-neurabin (amino acids 486–751) with HA- p70^S6k but not HA-AKT (Upper). Equal transfection of HA- p70^S6k and HA-AKT is confirmed by Western blot (Lower). Salt concentrations of washes are indicated. (c) Interaction of p70^S6k with neurabin is phosphorylation-independent. Coimmunoprecipitation of neurabin and p70^S6k is not affected by 24-h serum starvation, serum stimulation, or rapamycin treatment.

Figure 3

Tissue distribution of neurabin mRNA and protein in rat. (a) Northern blot of poly-A mRNA hybridized with radiolabeled neurabin probe. (b) Western blot probed with anti-neurabin antibody.

Figure 4

In situ hybridization of neurabin and p70^S6k mRNA. Sagittal rat brain sections showing (a) neurabin mRNA and (d) p70^S6k mRNA. Higher magnification of (b) neurabin and (e) p70^S6k in the hippocampus and (c) neurabin and (f) p70^S6k message in the cerebellum.

Figure 5

Neurabin and p70^S6k are found in the soluble fraction of synaptosomes from rat brain. Fractions are labeled as described in Materials and Methods: H, homogenate; P, pellet; and S, supernatant.

Figure 6

Model of p70^S6k regulation and targeting by neurabin. In nerve cell bodies, neurabin binds cytosolic p70^S6k. Subsequently, neurabin binds F-actin and is targeted to nerve terminals. p70^S6k kinase activity is enhanced by its interaction with neurabin, but its function at the synapse is unknown. Kinase activity may be regulated in response to mitogenic stimuli or possibly neuronal activity. (Inset) Neurabin oligomers bind the C terminus of p70^S6k via a single PDZ domain and bind F-actin with a unique N-terminal actin binding region. Oligomerization is potentially mediated by coiled–coiled structure at the C terminus of neurabin.