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Microtubule-associated protein 1B phosphorylation by glycogen synthase kinase 3ß is induced during PC12 cell differentiation

The MRC Centre for Developmental Neurobiology, New Hunts House, Guy’s Campus, King’s College London, London SE1 1UL, UK

View larger version (27K): [in a new window]   Fig. 1. MAP1B, MAP1B-P and GSK3ß levels during PC12 cell differentiation. (A) Immunoblot analysis of whole-cell extracts from PC12 cells treated with NGF from 0 to 5 days, showing the levels of MAP1B, MAP1B-P and GSK3ß during cell differentiation. Note MAP1B is not phosphorylated by GSK3ß prior to NGF treatment. (B) Time course of cell differentiation for PC12 cells treated with NGF from 0 to 5 days (defined as the percentage of cells bearing processes equal or greater in length than one cell diameter). (C) Average PC12 neurite length measured after zero, one, three and five DIV following NGF addition (B and C are means±s.e.m. from four independent experiments). (D-F) Quantitative immunoblot analysis of the levels of MAP1B (D), MAP1B-P (E) and GSK3ß (F) from PC12 cells treated with NGF from 0 to 5 days. Results are expressed as a proportion of the protein levels after five DIV and are means±s.e.m. from four independent experiments. MAP1B expression is closely related to cellular differentiation, whereas MAP1B-P expression is delayed by one to two days. GSK3ß levels change relatively little during differentiation, in contrast to the levels of MAP1B phosphorylation (i.e. MAP1B-P expression).

View larger version (90K): [in a new window]   Fig. 2. MAP1B-P expression is concentrated in extending neurites. (A) PC12 cells exposed to NGF for five days were fixed and stained with mAb SMI-31, which recognises MAP1B-P and a nuclear epitope unrelated to MAP1B (red), a MAP1B pAb (green) and YL1/2, a mAb specific for tyrosinated tubulin (blue), which stains all neurites and growth cones. MAP1B-P expression is concentrated in the longer neurites of the PC12 cells (arrowheads) and is undetectable in many shorter processes (arrows). By contrast, MAP1B is distributed throughout the cell, including the cell soma. (B) PC12 cells exposed to NGF for three days were fixed and stained with anti-GSK3ß antibodies (red), a MAP1B pAb (green) and YL1/2, a mAb specific for tyrosinated tubulin (blue). GSK3ß is expressed throughout the cell soma, neurites and growth cones.

View larger version (15K): [in a new window]   Fig. 3. Lithium inhibits neurite extension more effectively than differentiation. PC12 cells were treated with NGF in the presence of LiCl (2.5-10 mM) or NaCl (10 mM) or in control conditions. After three DIV, cultures were examined and the proportion of cells bearing one or more processes greater in length than one cell diameter (A) and the average neurite length in the field (B) were determined. At least 100 cells were analysed for each condition from each of four independent experiments. The levels of MAP1B and MAP1B-P in these cultures were determined by immunoblot analysis (D). Immunoblots were quantified and protein levels were normalised to controls. The levels of MAP1B-P are expressed as a proportion of MAP1B levels (C). Results are means±s.e.m. from four independent experiments. Note the close correlation between the Li+-induced inhibition of MAP1B phosphorylation and neurite extension.

View larger version (21K): [in a new window]   Fig. 4. GSK3ß activity increases during PC12 cell differentiation. (A) The GSK3ß activity in extracts from untreated PC12 cells and from cells exposed to NGF for five days was determined using a recombinant fragment of MAP1B (1B750) as a substrate. Phosphorylation was assayed by immunoblotting with mAb SMI-31 (1B750-P); recombinant protein was detected with anti-GST serum (1B750). Activity was virtually undetectable in unstimulated cells (DIV 0), but was readily detected five days after NGF addition (DIV 5). Inhibition of this activity by LiCl confirms that GSK3 is the kinase phosphorylating MAP1B in these cell extracts (DIV 5, + Li+). This increase in activity is not related to an increase in GSK3ß expression, which shows only a modest increase over the same period (GSK3ß). (B) GSK3ß activity towards recombinant MAP1B (1B750) in extracts from PC12 cells exposed to NGF from zero to five days was assayed as above. The level of phosphorylated 1B750 was divided by the level of GSK3ß, each determined by immunoblotting and normalised to five DIV, to give a measure of the activity of GSK3ß relative to its abundance during differentiation. Results are means±s.e.m. from four independent experiments. The increase in kinase activity induced by NGF reflects closely the level of endogenous MAP1B-P (Fig. 1).

View larger version (39K): [in a new window]   Fig. 5. Activation of GSK3ß by a soluble factor from differentiated PC12 cell extracts. (A) Equal amounts of protein from cells exposed to NGF for zero or five days were depleted of GSK3ß using specific antibodies (IPS, 0 DIV or 5 DIV). The extracts were collected and used in kinase assays alone or in conjunction with undepleted fractions from cells exposed to NGF for zero or five days (S1, 0 or 5 DIV). The in vitro kinase activity towards recombinant MAP1B present in the resultant samples was determined by immunoblotting with mAb SMI-31 (1B750-P); recombinant protein was detected with anti-GST serum (1B750). GSK3ß levels were determined by immunoblotting samples of the input fractions. Note the absence of kinase activity in differentiated cell extracts immunodepleted of GSK3ß (IPS (5 DIV)) and undifferentiated cell extracts (S1 (0 DIV)). Undifferentiated cell extracts mixed with immunodepleted differentiated cell extracts show high activity, but no significant increase in GSK3ß level. By contrast, immunodepleted undifferentiated cell extracts (IPS (0 DIV)) do not activate phosphorylation of MAP1B. (B) Prior phosphorylation of substrate (priming) does not activate MAP1B phosphorylation by GSK3ß. 1B750 was incubated with differentiated cell extracts immunodepleted of GSK3ß (IPS (5 DIV)) or with complete differentiated cell extracts in the presence of 20 mM LiCl for 16 hours at 37°C (S1 (5 DIV)/Li+) – conditions that inhibit phosphorylation of MAP1B by GSK3ß – and washed extensively in kinase buffer (at least 20 times the bead volume). The washed 1B750 was incubated with control buffer or with differentiated cell extracts (S1 (5 DIV)) or undifferentiated cell extracts (S1 (0 DIV)) for a further 16 hours at 37°C and kinase activity was then determined as in (A). Note that undifferentiated cell extracts show no kinase activity.

View larger version (30K): [in a new window]   Fig. 6. Post-translational modifications of GSK3ß during PC12 cell differentiation. Whole-cell extracts from PC12 cell cultures treated with NGF for the indicated times were immunoblotted with phosphospecific antibodies. (A) The levels of tyrosine-phosphorylated GSK3ß show a transient reduction following NGF treatment (Ptyr); changes in tyrosine phosphorylation correlate with the appearance of faster migrating bands reactive with a GSK3ß specific antibody (GSK3ß). Reduced phosphorylation is detectable by 15 minutes and has returned to unstimulated levels by three hours (Ptyr, lower band: note, the antibody used detects a conserved epitope present in both GSK3 and ß, hence the visualisation of a doublet on this blot). The levels of serine-phosphorylated GSK3ß (Pser) show a rapid increase within five minutes following NGF addition. Subsequently, phosphorylation gradually declines up to three hours, but does not return to baseline levels. (B) Longer-term changes in GSK3ß post-translational modifications were examined by immunoblotting. Overall levels of GSK3ß show a shallow rise following NGF addition (GSK3ß). This expression pattern is similar to that shown by tyrosine-phosphorylated GSK3ß. Indeed, the relative level of tyrosine-phosphorylated GSK3ß changes little when compared to total kinase over the five days of NGF treatment (Ptyr and D). NGF induces the expression of a band that migrates significantly more slowly on SDS gels than the GSK3ß present prior to NGF addition (GSK3ß and F). These two bands can be resolved more clearly on a 10% acrylamide gel (C). GSK3ß phosphorylation on serine-9 was readily detected in cells prior to NGF addition; note that these cultures had not been incubated in low serum medium prior to NGF addition. GSK3ß phosphorylation on serine-9 was maintained for two to three days after which it decreased to approximately half of the initial levels after five DIV (Pser and E). (D-F) quantitative immunoblot analysis of the relative levels of tyrosine- (D) or serine- (E) phosphorylated GSK3ß and the proportion of GSK3ß showing reduced electrophoretic mobility (F) from PC12 cells treated with NGF from 0 to 5 days. Results are means±s.e.m. from three independent experiments.