Proteomics analysis of the expression of neurogranin in murine neuroblastoma (Neuro-2a) cells reveals its involvement for cell differentiation

Abstract

Neurogranin (Ng) is a neural-specific, calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC). Although its biochemical property has been well characterized, the physiological function of Ng needs to be elucidated. In the present study, we performed proteomics analysis of the induced compositional changes due to the expression of Ng in murine neuroblastoma (Neuro-2a) cells using isotope coded affinity tags (ICAT) combined with 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS). We found that 40% of identified proteins were down-regulated and most of these proteins are microtubule components and associated proteins that mediated neurite outgrowth. Western blot experiments confirmed the expression of alpha-tubulin and microtubule- associated protein 1B (MAP 1B) was dramatically reduced in Neuro-2a-Ng cells compared to control. Cell morphology of Neuro-2a-Ng showed far less neurites than the control. Serum deprivation induced the extension of only one or two long neurites per cell in Neuro-2a-Ng, contrasting to the extension of multiple neurites per control cell. Ng may be linked to neurite formation by affecting expression of several microtubule related proteins. Furthermore, the PKC activator (PMA) induced an enhanced ERK1/2 activity in the cells that expressed Ng. The mutation of Ng at S36A caused sustained increase of ERK1/2 activity, whereas the ERK1/2 activity in mutation at I33Q showed no difference compared to wild type Ng, suggesting the phosphorylation of Ng but not the CaM /Ng interaction plays an important role in ERK activation. Ng may be involved in neuronal growth and differentiation via PKC and ERK1/2 signaling pathways.

Fig 1

Cytoskeletal protein expression was down- regulated in Neuro-2a-Ng cells. A, A typical μLC/MS spectrum of an ICAT-labeled sample. Proteins from Neuro-2a-Ng cells were labeled with heavy ¹³C reagent and those from Neuro-2a control were labeled with ¹²C reagent. The ¹³C/¹²C ratio of this ICAT labeled molecular ion pair was 0.4, representing the relative abundance of one peptide derived from Neuro-2a-Ng and Neuro-2a. B, Western blot validation of ICAT mass spectrometry results. The identities and corresponding ¹³C/¹²C ratios for the protein in this figure are as follows: MAP1B (0.3), α-tubulin (0.5). GAPDH is loading control. GAPDH was used as the loading control (5 μg of protein/lane).

Fig 2

Morphological effect of the expression of Ng in Neuro-2a cells. Neuro-2a-Ng displayed less differentiated appearance (B, E) compared to Neuro-2a control cells (A, D). After 24 hour serum deprivation, in contrast to the multipolar type of neurite growth observed in Neuro-2a control cells, a large number of Neuro-2a-Ng cells have a distinct differentiated morphology in which cell bodies extend only one or two long neurites that are generally unbranched. C and F are statistical analysis for neurite-bearing cells in both Neuro-2a and Neuro-2a-Ng without or with serum starvation, respectively. *p < 0.05 and **p < 0.001.

Fig 3

The expression of Ng induced sustained activation and up-regulation of ERK1/2 phosphorylation by PMA treatment in Neuro-2a-Ng cells. The time points after the treatment of 300 nM PMA were shown above. The phosphorylation of ERK1/2, total ERK1/2 and Ng expression level were detected by Western blot analysis (10 μg of protein/lane) (A). The quantitation of Western blot images of the ratio between phospho-ERK1/2 and total-ERK was shown in B. *p < 0.05 and **p < 0.001, comparing Neuro-2a-Ng to Neuro-2a cells (n = 3).

Fig 4

Phosphorylation of ERK1/2 after 300 nM PMA treatment in HEK293 transiently transfected with EGFP or EGFP-Ng wild-type. The time points after the treatment of 300 nM PMA were shown above. The phosphorylation of ERK1/2, total ERK1/2, Ng expression level and the phosphorylation of Ng were detected by Western blot analysis (10 μg of protein/lane) (A). The quantitation of Western blot images of the ratio between phospho-ERK1/2 and total-ERK was shown in B. *p < 0.05 and **p < 0.001, comparing wild type HEK293 to HEK293 cells transfected with Ng (n = 4).

Fig 5

The ERK phosphorylation induced by PMA is mediated by PKC. A, The pre-incubation with PKC specific inhibitor, 5 μM GF102903X, totally abolished the ERK1/2 phosphorylation induced by 300 nM PMA in HEK 293 transfected with Ng wild type. B, Western blot analysis of ERK1/2 phosphorylation after 300 nM PMA treatment in HEK 293 transfected with Ng wild type, S36A and I33Q mutants. The time points after the treatment of PMA were shown above.