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First published online February 23, 2005
doi: 10.1242/10.1242/jcs.01697

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Glycogen synthase kinase-3ß phosphorylation of MAP1B at Ser1260 and Thr1265 is spatially restricted to growing axons

Niraj Trivedi^1,*, Phil Marsh², Robert G. Goold¹, Alison Wood-Kaczmar¹ and Phillip R. Gordon-Weeks^1,

¹ The MRC Centre for Developmental Neurobiology, New Hunts House, Guy's Campus, King's College London, London SE1 1UL, UK
² Molecular Biology Unit, New Hunts House, Guy's Campus, King's College London, London SE1 1UL, UK

View larger version (51K): [in a new window]   Fig. 1. The recombinant MAP1B protein SP contains the GSK-3ß phosphorylation site recognised by mAb SMI-31, whereas SP does not. (A) Diagram showing full-length mouse MAP1B and the two MAP1B recombinant proteins (SP and SP) used in the kinase assay. The SPAKS site is shown as a hatched box and its sequence, beneath the full-length MAP1B, in the single letter amino acid code. One recombinant protein (SP) encodes amino acids 1244-1530, and hence starts at the beginning of the SPAKS sequence, whereas the other (SP) lacks the SPAKS sequence by starting at amino acid 1264. GST, glutathione S-transferase. (B) Western immunoblots of SP and SP GST-pull downs that were incubated with (+), or without (–), a high-speed supernatant from neonatal rat brain (S1) in kinase buffer showed that SP was positive for the mAb SMI-31 epitope, whereas SP was negative. The same blot was then stripped and re-probed with an anti-GST antibody (GST) to assess the protein loading levels, which were found to be similar (lower panel). Note that both recombinants, when incubated with S1, show an upward band shift. This is normally associated with protein phosphorylation, and suggests, therefore, that the SP recombinant becomes phosphorylated in the kinase assay, but at a site or sites not recognised by mAb SMI-31.

View larger version (31K): [in a new window]   Fig. 2. GSK-3ß phosphorylates MAP1B on Ser1260. The serine residues at positions 1247, 1251, 1255, 1257 and 1260 (mouse sequence) were replaced by valine residues to produce recombinant proteins SP1 to SP5, respectively. Kinase assays were performed using the recombinant proteins as substrates. Recombinant proteins were incubated with a high-speed supernatant from neonatal rat brain (S1) in kinase buffer to investigate if they could be phosphorylated by GSK-3ß at the site recognised by the mAb SMI-31. Western immunoblot analysis of the kinase assay GST pull-downs showed that recombinants SP1 to SP4 were positive for the mAb SMI-31 epitope. However, the expression of the mAb SMI-31 epitope by the SP5 recombinant incubated with S1 was greatly reduced in comparison to the other recombinants, including SP (c.f. Fig. 1). This indicates that the serine at position 1260 in the full-length mouse MAP1B sequence is phosphorylated by GSK-3ß to produce the mAb SMI-31 epitope. Controls (Con) were treated in the same way but without the inclusion of S1. The same blot was then stripped and reprobed with an anti-GST antibody (GST) to assess the protein loading levels, which were found to be similar (lower panel).

View larger version (39K): [in a new window]   Fig. 3. The effects of double point mutations on GSK-3ß phosphorylation of MAP1B. To produce double point mutated recombinant proteins SP1/5, SP2/5, SPS/5, SP3/5, SP4/5 and SPT/5, the serine residues at positions 1247, 1251, 1253, 1255 and 1257 and Thr1265, respectively, were replaced by valine residues in the SP recombinant in which Ser1260 was replaced by a valine (see Table 1). Kinase assays were performed using the recombinant proteins as substrates. Recombinant proteins were incubated with a high-speed supernatant from neonatal rat brain (S1) in kinase buffer to investigate if they could be phosphorylated by GSK-3ß at the site recognised by the mAb SMI-31. Recombinant proteins SP1/5, SP2/5, SP3/5 and SPS/5 were immunoreactive with mAb SMI-31 after incubation in the kinase assay as was SP5 itself. However, recombinant proteins SP4/5 and SPT/5 were not immunoreactive, or showed greatly reduced immunoreactivity, with mAb SMI-31. To enhance the relatively weak mAb SMI-31 signal of the SP5 recombinant protein the blots were overexposed, as can be seen from the SP control (c.f. Fig. 2). The same blot was then stripped and reprobed with an anti-GST antibody (GST) to assess the protein loading levels, which were similar (lower panel).

View larger version (58K): [in a new window]   Fig. 4. Polyclonal antibodies BUGS and SuperBUGS recognise phosphorylated Ser1260 and phosphorylated Thr1265 in MAP1B respectively. Western immunoblot analysis of SP, SPT, SP5 and SPT/5 recombinant proteins that were incubated with a high-speed supernatant from neonatal rat brain (S1) in kinase buffer to investigate if they could be phosphorylated at the sites recognised by mAb SMI-31, pAb BUGS and pAb SuperBUGS. All three antibodies recognised native MAP1B in the S1 supernatant (arrowheads) and the SP recombinant protein following phosphorylation in the kinase assay. The recombinant protein SPT was not recognised by mAb SMI-31 or by pAb SuperBUGS but this mutation did not affect the binding of pAb BUGS, suggesting that the S1265V mutation did not affect GSK-3ß phosphorylation of Ser1260. In confirmation of earlier results, the SP5 recombinant showed a small, residual binding of mAb SMI-31. As expected, pAb BUGS did not recognise the SP5 recombinant protein whereas pAb SuperBUGS did. The double point-mutated recombinant protein SPT/5 was not recognised by any of the three antibodies. The same blot was stripped and reprobed with an anti-GST antibody (GST) to assess the protein loading levels, which were similar.

View larger version (37K): [in a new window]   Fig. 5. The phosphorylation sites recognised by pAb BUGS and pAb SuperBUGS are generated by GSK-3ß phosphorylation. Western immunoblot analysis of the SP recombinant protein incubated with a high-speed supernatant from neonatal rat brain (S1) in kinase buffer (+). Pre-treatment of S1 with 20 mM lithium chloride (Li) considerably reduced the binding of pAb BUGS and pAb SuperBUGS to the SP recombinant suggesting that GSK-3ß phosphorylation is necessary for these antibodies to recognise the SP protein. 20 mM sodium chloride (Na) was added to control for possible ion and concentration effects of lithium chloride. The same blots were then stripped and re-probed with an anti-GST antibody (GST) to assess the protein loading levels, which were similar. Note that band-shifts of SP produced by S1 incubation are reduced by lithium but not abolished, suggesting that SP is additionally phosphorylated by kinases other than GSK-3ß. The last track shows that baculovirus produced recombinant GSK-3ß can phosphorylate SP at the pAb BUGS and the pAb SuperBUGS site.

View larger version (43K): [in a new window]   Fig. 6. Polyclonal antibody BUGS and pAb SuperBUGS recognise native MAP1B in E12 rat embryos. Western immunoblots of tissue from an E12 rat embryo probed with antibodies against MAP1B (mAb AA6) and GSK-3ß phosphorylated MAP1B (mAb SMI-31, pAb BUGS and pAb SuperBUGS). Monoclonal antibody AA6 recognises a single, high molecular weight protein band corresponding to MAP1B. Monoclonal antibody SMI-31 also recognises MAP1B but, in addition, several lower molecular weight protein bands probably corresponding to neurofilament and nuclear epitopes (Lichtenberg-Kraag et al., 1992). Polyclonal antibody BUGS and pAb SuperBUGS recognise a single protein band that co-migrates with MAP1B (arrowhead). Molecular weights (kDa) are indicated on the left hand side.

View larger version (175K): [in a new window]   Fig. 7. The expression of GSK-3ß-phosphorylated MAP1B is restricted to growing axons in rat embryos. Light micrographs of transverse sections of E12 rat embryo spinal cords. (A) Polyclonal antibody BUGS stains growing axons within the spinal cord, including commissural axons (arrows) and axons of the medial longitudinal tract (mlt), and axons within the PNS, including the axons of motor neurons (curved arrow) and primary sensory neurons (arrowheads). However, the proximal regions of these axons and their parent cell bodies are unstained. For example, the axonal staining in the dorsal root ganglia is largely restricted to the poles of the ganglion whereas the central regions of the ganglia, where proximal axons and cell bodies are located, are unstained (asterisk; compare with C). (B) Polyclonal antibody SuperBUGS has a staining pattern similar to that of pAb BUGS (A) except that the lack of staining of the proximal regions of axons is more pronounced. For example there is an unstained gap between the spinal cord and the proximal staining of axons in the ventral roots (arrowheads with asterisks). SuperBUGS also stains the spindle apparatus of mitotic cells scattered throughout the embryo but in particularly high numbers in the ventricular zone (arrowheads) near the central canal (cc). The inset shows a high power view of the staining of mitotic cells. (C) Polyclonal antibody MAP1B N-19 recognises all forms of MAP1B. Neurons are stained throughout their entirety including cell bodies and axons. Neuronal cell bodies in the dorsal root ganglia (asterisk) and the ventral motor neuron pool (mn) are particularly prominent. (D) Polyclonal antibody to GSK-3ß stains neurons entirely including cell bodies and axons. Neuronal cell bodies in the dorsal root ganglia (asterisk) and the ventral motor neuron pool (mn) are particularly prominent. Bar, 100 µm.

View larger version (36K): [in a new window]   Fig. 8. Polyclonal antibody SuperBUGS labels axons in a gradient that is highest towards the growth cone in cultured cerebral cortical neurons. (A-C) Low power, confocal fluorescence images of cerebral cortical cultures from an E16 mouse labelled with phalloidin (A), which labels actin filaments, and immunolabelled with a phosphate-independent MAP1B antibody (B) and pAb SuperBUGS (C). Despite the distribution of MAP1B throughout the neuron, pAb SuperBUGS only labels the axon (minor processes are unlabelled) and in a gradient that is highest towards the growth cone. (D-F) High power, confocal fluorescence images of a growth cone from an E16 mouse cerebral cortical culture labelled with phalloidin (D), which labels actin filaments, and immunolabelled with an antibody against tubulin (E) and pAb SuperBUGS (F). Polyclonal antibody SuperBUGS partly colocalises with microtubules but also extends into the P-domain of the growth cone where the filamentous actin is concentrated. (G-I) High power, confocal fluorescence images of a growth cone from an E16 mouse cerebral cortical culture labelled with phalloidin (G), which labels actin filaments, and immunolabelled with an antibody against tubulin (H) and pAb BUGS (I). Polyclonal antibody BUGS partly colocalises with microtubules but also extends into the P-domain of the growth cone, where the filamentous actin is concentrated. Bar, 40 µm (A-C); 5 µm (D-I).

View larger version (52K): [in a new window]   Fig. 9. Co-transfection of COS-7 cells with GSK-3ß and MAP1B, mutated at Ser1260, Thr1265 or both, partially rescues the loss of stable microtubules. (A-F) Low power, confocal fluorescence images of COS-7 cells transfected with GSK-3ß and wild-type MAP1B and immunolabelled with antibodies against GSK-3ß (A,D), a goat polyclonal antibody that recognises all forms of MAP1B (B,E) and either pAb SuperBUGS (C) or pAb BUGS (F). Cells expressing both MAP1B and GSK-3ß are immunopositive for polyclonal antibodies SuperBUGS and BUGS. Note the localisation of GSK-3ß in the nucleus as well as the cytoplasm of transfected cells (asterisk in A and D). (G-L) Low power, confocal fluorescence images of COS-7 cells transfected with GSK-3ß and either wild-type MAP1B (G-I) or MAP1B in which Ser1260 had been replaced by valine (J-L) and immunolabelled with antibodies against: GSK-3ß (G,J), a goat polyclonal antibody that recognises all forms of MAP1B (H,K), and pAb SUP GLU, which recognises stable microtubules (I,L). In cells expressing GSK-3ß and wild-type MAP1B there is a complete loss of stable microtubules (G-I) whereas in cells expressing GSK-3ß and mutated MAP1B, in which Ser1260 has been replaced by valine, a few stable microtubules remain (arrowheads). In non-transfected cells, the numbers of stable microtubules are high (asterisks in I and L). Note the localisation of GSK-3ß in the nucleus as well as the cytoplasm of transfected cells (asterisk in G and J). (M) Histogram showing the concentration of pAb SUP GLU fluorescence in COS-7 cells transfected with wild-type MAP1B (WT), MAP1B in which Ser1260 has been mutated to valine (S1260V), MAP1B in which Thr1265 has been mutated to valine (T1265V) or MAP1B in which both Ser1260 and Thr1265 have been mutated to valine (Double). Transfection with mutated MAP1B partially rescues the loss of stable microtubules seen with wild-type MAP1B. For each bar, the values are the mean±s.e.m. of results from ten cells from three separate experiments. Bar, 10 µm.

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