Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 9

1.
Figure 9

Figure 9. Sequence alignment of MSK and RSK around phosphorylation sites. From: MSK1 activity is controlled by multiple phosphorylation sites.

The sequences around the T-loop, linker and C-terminal phosphorylation sites of MSK were aligned with RSK. Phosphorylation sites are highlighted in black and conserved residues are highlighted in grey. The hydrophobic motif in RSK is underlined and the MAPK docking site is indicated by asterisks. Black ‘lollipops’ are phosphorylation sites that are conserved between MSK and RSK isoforms. White ‘lollipops’ are phosphorylation sites that are unique to MSK1.

Claire E. McCOY, et al. Biochem J. 2005 April 15;387(Pt 2):507-517.
2.
Figure 1

Figure 1. Autophosphorylation of MSK1 in vitro. From: MSK1 activity is controlled by multiple phosphorylation sites.

Recombinant MSK1, which had been previously phosphorylated with ERK2, was autophosphorylated in vitro as described in the Methods section. The autophosphorylated MSK1 was then run on an SDS/polyacrylamide gel and the MSK1 band excised, digested with trypsin, and the resulting peptides were run on reversed-phase HPLC as described in the Methods section. The 32P elution trace is shown in (A). Phosphopeptides eluted in the 32P-labelled fractions were identified by MS, and the positions of phosphate in the peptides were determined by solid-phase sequencing for each of the peaks (BF).

Claire E. McCOY, et al. Biochem J. 2005 April 15;387(Pt 2):507-517.
3.
Figure 8

Figure 8. C-terminal phosphorylation of MSK1 does not affect its interaction with MAPKs. From: MSK1 activity is controlled by multiple phosphorylation sites.

FLAG-MSK1 (wild-type, Leu740→Ala, Arg742→Ala/Arg43→Ala, Ser750→Ala/Ser752→Ala or Ser758→Ala) was transfected into HEK-293 cells. Cells were serum-starved for 16 h and then stimulated with UV-C (200 J/m2, followed by 30 min at 37 °C) or PMA (400 ng/ml, 10 min) as indicated and lysed as described in the Methods section. MSK1 was immunoprecipitated using an anti-FLAG antibody and immunoprecipitates were run on SDS gels. Gels were immunoblotted for FLAG, ERK1, ERK2 or p38α.

Claire E. McCOY, et al. Biochem J. 2005 April 15;387(Pt 2):507-517.
4.
Figure 2

Figure 2. Phosphorylation of MSK1 in HEK-293 cells. From: MSK1 activity is controlled by multiple phosphorylation sites.

(A) Wild-type (WT), C-terminal kinase dead (CT-KD) or N-terminal kinase dead (NT-KD) GST-tagged MSK1 was expressed in HEK-293 cells by transient transfection. Cells were serum-starved for 16 h and incubated with PD184352 (2 μM) or SB203580 (5 μM) where indicated. Cells were then stimulated with UV-C (200 J/m2, followed by 30 min at 37 °C) or PMA (TPA; 400 ng/ml, 10 min) as indicated and lysed as described in the Methods section. Lysates were immunoblotted using antibodies against GST, or MSK1 phosphorylated on Ser-212, Ser-360, Ser-376, Ser-381, Thr-581, Ser-750 or dual phosphorylated Ser-750/Ser-752. (B) Endogenous MSK1 was immunoprecipitated (IP) from HEK-293 cells and treated as indicated. Immunoprecipitates were run on SDS gels and blotted for total MSK1, phospho-Ser-212, Ser-360, Ser-376, Ser-381, Thr-581, Ser-750 and dual phosphorylated Ser-750/Ser-752.

Claire E. McCOY, et al. Biochem J. 2005 April 15;387(Pt 2):507-517.
5.
Figure 6

Figure 6. Phosphorylation of Ser-381 is not essential for activity. From: MSK1 activity is controlled by multiple phosphorylation sites.

Wild-type or Ser381→Ala FLAG-tagged MSK1 was expressed in HEK-293 cells by transient transfection. Cells were serum-starved for 16 h and then stimulated with UV-C (200 J/m2, followed by 30 min at 37 °C) or PMA (400 ng/ml, 10 min) as indicated and lysed as described in the Methods section. MSK1 was immunoprecipitated using an anti-FLAG antibody and assayed against a peptide substrate as described in the Methods section. One unit (U) of activity was defined as the amount of kinase required to incorporate 1 nmol of phosphate in 1 min. The kinase activity from unstimulated cells is shown by open bars, UV-C stimulation as hatched bars and PMA stimulation as grey bars. Error bars represent the S.D. for three assays. The same lysates were also immunoblotted with antibodies against FLAG, phospho-Ser-360 MSK1, phospho-Ser-376 MSK1 or phospho-Thr-581 MSK1.

Claire E. McCOY, et al. Biochem J. 2005 April 15;387(Pt 2):507-517.
6.
Figure 3

Figure 3. Ser-376 and Thr-581 phosphorylation is required for MSK1 activity. From: MSK1 activity is controlled by multiple phosphorylation sites.

Wild-type, Ser360→Ala, Ser376→Ala or Thr581→Ala FLAG-tagged MSK1 was expressed in HEK-293 cells by transient transfection. Cells were serum-starved for 16 h and then stimulated with UV-C (200 J/m2, followed by 30 min at 37 °C) or PMA (TPA; 400 ng/ml, 10 min) as indicated and lysed as described in the Methods section. MSK1 was immunoprecipitated using an anti-FLAG antibody and assayed against a peptide substrate as described in the Methods section. The kinase activity from unstimulated cells is shown by open bars, UV-C stimulation as hatched bars and PMA stimulation as grey bars. One unit (U) of activity is defined as the amount of kinase required to incorporate 1 nmol of phosphate in 1 min. Error bars represent the S.D. for three assays. The same lysates were also immunoblotted with antibodies against FLAG or MSK1 phosphorylated on Ser-212, Ser-360, Ser-376, Ser-381 or Thr-581.

Claire E. McCOY, et al. Biochem J. 2005 April 15;387(Pt 2):507-517.
7.
Figure 4

Figure 4. MSK1 autophosphorylates on Ser-212 in HEK-293 cells. From: MSK1 activity is controlled by multiple phosphorylation sites.

(A) Wild-type MSK1 or Ser212→Ala FLAG-tagged MSK1 was expressed in HEK-293 cells by transient transfection. Cells were serum-starved for 16 h and then stimulated with UV-C (200 J/m2, followed by 30 min at 37 °C) or with PMA (400 ng/ml, 10 min) as indicated and lysed as described in the Methods section. MSK1 was immunoprecipitated using an anti-FLAG antibody and assayed against a peptide substrate as described in the Methods section. One unit (U) of activity is defined as the amount of kinase required to incorporate 1 nmol of phosphate in 1 min. The kinase activity from unstimulated cells is shown by open bars, UV-C stimulation as hatched bars and PMA stimulation as grey bars. Error bars represent the S.D. for three assays. The same lysates were also immunoblotted with antibodies against FLAG or MSK1 phosphorylated on Ser-212, Ser-360, Ser-376, Ser-381 or Thr-581. (B) Same as (A) except that GST wild-type (WT), C-terminal kinase dead (CT-KD), Ser376→Asp or C-terminal kinase dead/Ser376→Asp were expressed in HEK-293 cells. MSK1 was pulled down from 0.2 mg of lysate with glutathione–Sepharose and assayed against a peptide substrate as described in the Methods section. The same lysates were also immunoblotted with antibodies against FLAG or MSK1 phosphorylated on either Ser-212 or Ser-381.

Claire E. McCOY, et al. Biochem J. 2005 April 15;387(Pt 2):507-517.
8.
Figure 5

Figure 5. Phosphorylation of Ser-212 of MSK1 in HEK-293 cells. From: MSK1 activity is controlled by multiple phosphorylation sites.

MS of the tryptic phosphopeptide (210–226 of MSK1) containing Ser-212. MSK1 [GST wild-type (A, E), C-terminal kinase dead (B, F), Ser376→Asp (C, G) or C-terminal kinase dead/Ser376→Asp (D, H)] was expressed in HEK-293 cells (control samples, AD respectively) and from a similar set of PMA-stimulated cells (+PMA samples, EH respectively). MSK1 was pulled down from 15 mg of lysate with glutathione–Sepharose and run on a 4–12% acrylamide/SDS gel. Bands corresponding to MSK1 were excised, reduced, alkylated with iodoacetamide and then digested or not with trypsin [37]. The resultant tryptic phosphopeptides were subjected to immobilized metal affinity chromatography as described in the Methods section, followed by MS on an Applied Biosystems 4700 MALDI–TOF-TOF with 0.25 μl of α-cyanocinnamic acid+10 mM NH4PO3 as the matrix. The theoretical monoisotopic mass for the peptide 210–226 (AYSFCGTIEYMAPDIVR) with one phosphoserine is 2072.89 Da, and with one phosphoserine and one oxidized methionine residue is 2088.88 Da (the cysteine residue having been converted into carbamidomethylcysteine).

Claire E. McCOY, et al. Biochem J. 2005 April 15;387(Pt 2):507-517.
9.
Figure 7

Figure 7. MSK1 requires a MAPK docking site for activation. From: MSK1 activity is controlled by multiple phosphorylation sites.

(A) Wild-type, Leu740→Ala or Arg743→Ala/Arg744→Ala FLAG-tagged MSK1 was expressed in HEK-293 cells by transient transfection. Cells were serum-starved for 16 h and then stimulated with UV-C (200 J/m2, followed by 30 min at 37 °C) or PMA (400 ng/ml, 10 min) as indicated and lysed as described in the Methods section. MSK1 was immunoprecipitated using an anti-FLAG antibody and assayed against a peptide substrate as described in the Methods section. One unit (U) of activity was defined as the amount of kinase required to incorporate 1 nmol of phosphate in 1 min. The kinase activity from unstimulated cells is shown by open bars, UV-C stimulation as hatched bars and PMA stimulation as grey bars. Error bars represent the S.D. for three data points. The same lysates were also immunoblotted with antibodies against FLAG, or MSK1 phosphorylated on Ser-212, Ser-360, Ser-376, Ser-381, Thr-581, Ser-750 or dual phosphorylated Ser-750/Ser-752. (B) Same as (A) except that wild-type, Ser750→Ala/Ser752→Ala or Ser758→Ala was expressed in HEK-293 cells.

Claire E. McCOY, et al. Biochem J. 2005 April 15;387(Pt 2):507-517.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk