Localisation of Microtubule‐Associated Protein 1B Phosphorylation Sites Recognised by Monoclonal Antibody SMI‐31

Johnstone, Mandy; Goold, Robert; Bei, Di; Fischer, Itzhak; Gordon‐Weeks, Phillip R.

doi:10.1046/j.1471-4159.1997.69041417.x

Cited by 34 publications

(39 citation statements)

References 30 publications

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“…1) -corresponding to mouse MAP1B Journal of Cell Science 122 (14) amino acids 1244-1530, and therefore containing the S1388 and S1392 sites (Trivedi et al, 2005) -only after it has been phosphorylated in vitro with a neonatal rat brain extract containing MAP1B kinases (Fig. 2C) (Johnstone et al, 1997). We have previously shown that this is also true of SuperBUGS and BUGS (Trivedi et al, 2005).…”

Section: Production and Characterisation Of Phosphospecific Antibodiementioning

confidence: 79%

“…We purified MAP1B from neonatal rat brain (Johnstone et al, 1997) and mapped the phosphorylation sites by mass spectrometry. This MAP1B is a developmentally regulated microtubule-associated phosphoprotein that regulates microtubule dynamics in growing axons and growth cones.…”

Section: A Comparison Of the Phosphorylation Maps Of Juvenile And Adumentioning

confidence: 99%

“…(A) Affinity-purified pAbs against mouse MAP1B S1260-P (BUGS) and S1265-P (SuperBUGS), and against rat S1388-P and S1392-P were immunoblotted against proteins from neonatal rat whole brain (W) or neonatal rat brain supernatant (S1). All of these antibodies recognise a protein that co-migrates with MAP1B (~320 kDa), as indicated by comparison with protein bands recognised by pAb anti-MAP1B-C1, which recognises all forms of MAP1B (Johnstone et al, 1997). (B) Phospho-peptide inhibition assay.…”

Section: Embryonic-spinal-cord Expression Of Phosphorylated Map1bmentioning

confidence: 99%

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Nonprimed and DYRK1A-primed GSK3β-phosphorylation sites on MAP1B regulate microtubule dynamics in growing axons

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Lin

Kraus

et al. 2009

Journal of Cell Science

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114

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MAP1B is a developmentally regulated microtubule-associated phosphoprotein that regulates microtubule dynamics in growing axons and growth cones. We used mass spectrometry to map 28 phosphorylation sites on MAP1B, and selected for further study a putative primed GSK3β site and compared it with two nonprimed GSK3β sites that we had previously characterised. We raised a panel of phosphospecific antibodies to these sites on MAP1B and used it to assess the distribution of phosphorylated MAP1B in the developing nervous system. This showed that the nonprimed sites are restricted to growing axons, whereas the primed sites are also expressed in the neuronal cell body. To identify kinases phosphorylating MAP1B, we added kinase inhibitors to cultured embryonic cortical neurons and monitored MAP1B phosphorylation with our panel of phosphospecific antibodies. These experiments identified dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK1A) as the kinase that primes sites of GSK3β phosphorylation in MAP1B, and we confirmed this by knocking down DYRK1A in cultured embryonic cortical neurons by using shRNA. DYRK1A knockdown compromised neuritogenesis and was associated with alterations in microtubule stability. These experiments demonstrate that MAP1B has DYRK1A-primed and nonprimed GSK3β sites that are involved in the regulation of microtubule stability in growing axons.

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Section: Production and Characterisation Of Phosphospecific Antibodiementioning

confidence: 79%

Section: A Comparison Of the Phosphorylation Maps Of Juvenile And Adumentioning

confidence: 99%

Section: Embryonic-spinal-cord Expression Of Phosphorylated Map1bmentioning

confidence: 99%

See 1 more Smart Citation

Nonprimed and DYRK1A-primed GSK3β-phosphorylation sites on MAP1B regulate microtubule dynamics in growing axons

Scales

Lin

Kraus

et al. 2009

Journal of Cell Science

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114

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“…5B and data not shown). We used an antibody that recognizes the total amount of MAP1B, independently of its phosphorylation state (Fischer and Romano-Clarke, 1990), and specific antibodies that distinguish the two types of MAP1B phosphorylation: the SMI31 monoclonal antibody for mode I (Johnstone et al, 1997), and the 125 monoclonal antibody specific to mode II (Ulloa et al, 1993). The protein levels of all MAP1B forms were reduced in the COUP-TFI null brains (Fig.…”

Section: Research Articlementioning

confidence: 99%

COUP-TFI is required for the formation of commissural projections in the forebrain by regulating axonal growth

et al. 2006

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The transcription factor COUP-TFI (NR2F1), an orphan member of the nuclear receptor superfamily, is an important regulator of neurogenesis, cellular differentiation and cell migration. In the forebrain, COUP-TFI controls the connectivity between thalamus and cortex and neuronal tangential migration in the basal telencephalon. Here, we show that COUP-TFI is required for proper axonal growth and guidance of all major forebrain commissures. Fibres of the corpus callosum, the hippocampal commissure and the anterior commissure project aberrantly and fail to cross the midline in COUP-TFI null mutants. Moreover, hippocampal neurons lacking COUP-TFI have a defect in neurite outgrowth and show an abnormal axonal morphology. To search for downstream effectors, we used microarray analysis and showed that, in the absence of COUP-TFI, expression of various cytoskeleton molecules involved in neuronal morphogenesis is affected. Diminished protein levels of the microtubule-associated protein MAP1B and increased levels of the GTP-binding protein RND2 were confirmed in the developing cortex in vivo and in primary hippocampal neurons in vitro. Therefore, based on morphological studies, gene expression profiling and primary cultured neurons, the present data uncover a previously unappreciated intrinsic role for COUP-TFI in axonal growth in vivo and supply one of the premises for COUP-TFI coordination of neuronal morphogenesis in the developing forebrain.

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“…Lysates were assayed for GSK3b activity using a recombinant fragment of MAP1B (1B750) as a substrate (Johnstone et al 1997;Lucas et al 1998;Goold and Gordon-Weeks 2001). Extracts from cultures not treated with NGF were used as a negative control.…”

Section: Measurement Of Gsk3b Activitymentioning

confidence: 99%

NGF activates the phosphorylation of MAP1B by GSK3β through the TrkA receptor and not the p75^NTRreceptor

Goold

Gordon‐Weeks

2003

Journal of Neurochemistry

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We have recently shown that nerve growth factor (NGF) induces the phosphorylation of the microtubule-associated protein 1B (MAP1B) by activating the serine/threonine kinase glycogen synthase kinase 3b (GSK3b) in a spatio-temporal pattern in PC12 cells that correlates tightly with neurite growth. PC12 cells express two types of membrane receptor for NGF: TrkA receptors and p75 NTR receptors, and it was not clear from our studies which receptor was responsible. We show here that brain-derived neurotrophic factor, which activates p75 NTR but not TrkA receptors, does not stimulate GSK3b phosphorylation of MAP1B in PC12 cells. Similarly, NGF fails to activate GSK3b phosphorylation of MAP1B in PC12 cells that lack TrkA receptors but express p75 NTR receptors (PC12 nnr). Chick ciliary ganglion neurons in culture lack TrkA receptors but express p75 NTR and also fail to show NGF-dependent GSK3b phosphorylation of MAP1B, whereas in rat superior cervical ganglion neurons in culture, NGF activation of TrkA receptors elicits GSK3b phosphorylation of MAP1B. Finally, inhibition of TrkA receptor tyrosine kinase activity in PC12 cells and superior cervical ganglion neurons with K252a potently and dose-dependently inhibits neurite elongation while concomitantly blocking GSK3b phosphorylation of MAP1B. These results suggest that the activation of GSK3b by NGF is mediated through the TrkA tyrosine kinase receptor and not through p75 NTR receptors. Keywords: glycogen synthase kinase 3b, microtubule-associated protein 1B, nerve growth factor, PC12 cells. Glycogen synthase kinase 3 (GSK3) is a highly conserved serine/threonine kinase, originally identified as a negative regulator of glycogen synthase, but more recently implicated in a wide range of signalling pathways, including ones influencing cell fate determination during embryogenesis and neuronal differentiation (Grimes and Jope 2001;Doble and Woodgett 2003). In vertebrates, there are two isoforms, GSK3a and GSK3b, encoded by separate genes (Woodgett 1990) and, although GSK3a is the dominant form in most tissues, GSK3b is particularly abundant in neural tissue (Woodgett 1990), where it is neuron-specific (Takahashi et al. 1994;Leroy and Brion 1999). In the CNS, GSK3b is developmentally regulated, with peak levels of expression during axonogenesis. It is present in growing axons but completely excluded from them at the end of axonogenesis, being restricted in the adult to neuronal cell bodies and dendrites (Leroy and Brion 1999). This suggests that GSK3b has a role in axonogenesis, an idea supported by pharmacological studies. GSK3 is inhibited by lithium ions (Klein and Melton 1996;Stambolic et al. 1996) and although other enzymes are also inhibited by lithium, in immature neurons, GSK3 appears to be the primary pharmacological target (Lucas et al. 1998;Hall et al. 2002; Owen and GordonWeeks 2003; but see Williams et al. 2002). Lithium has profound morphological effects on developing neurons in culture (Burstein et al. 1985;Hollander and Bennett 1991;Lucas and Salinas 19...

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Localisation of Microtubule‐Associated Protein 1B Phosphorylation Sites Recognised by Monoclonal Antibody SMI‐31

Cited by 34 publications

References 30 publications

Nonprimed and DYRK1A-primed GSK3β-phosphorylation sites on MAP1B regulate microtubule dynamics in growing axons

Nonprimed and DYRK1A-primed GSK3β-phosphorylation sites on MAP1B regulate microtubule dynamics in growing axons

COUP-TFI is required for the formation of commissural projections in the forebrain by regulating axonal growth

NGF activates the phosphorylation of MAP1B by GSK3β through the TrkA receptor and not the p75^NTRreceptor

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Localisation of Microtubule‐Associated Protein 1B Phosphorylation Sites Recognised by Monoclonal Antibody SMI‐31

Cited by 34 publications

References 30 publications

Nonprimed and DYRK1A-primed GSK3β-phosphorylation sites on MAP1B regulate microtubule dynamics in growing axons

Nonprimed and DYRK1A-primed GSK3β-phosphorylation sites on MAP1B regulate microtubule dynamics in growing axons

COUP-TFI is required for the formation of commissural projections in the forebrain by regulating axonal growth

NGF activates the phosphorylation of MAP1B by GSK3β through the TrkA receptor and not the p75NTRreceptor

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NGF activates the phosphorylation of MAP1B by GSK3β through the TrkA receptor and not the p75^NTRreceptor