CDK-mediated regulation of cell functions via c-Jun phosphorylation and AP-1 activation

Abstract

Cyclin-dependent kinases (CDKs) and their targets have been primarily associated with regulation of cell-cycle progression. Here we identify c-Jun, a transcription factor involved in the regulation of a broad spectrum of cellular functions, as a newly recognized CDK substrate. Using immune cells from mouse and human, and several complementary in vitro and in vivo approaches including dominant negative protein expression, pharmacologic inhibitors, kinase assays and CDK4 deficient cells, we demonstrate the ability of CDK4 to phosphorylate c-Jun. Additionally, the activity of AP-1, a ubiquitous transcription factor containing phosphorylated c-Jun as a subunit, was inhibited by abrogating CDK4. Surprisingly, the regulation of c-Jun phosphorylation by CDK4 occurred in non-dividing cells, indicating that this pathway is utilized for cell functions that are independent of proliferation. Our studies identify a new substrate for CDK4 and suggest a mechanism by which CDKs can regulate multiple cellular activation functions, not all of which are directly associated with cell cycle progression. These findings point to additional roles of CDKs in cell signaling and reveal potential implications for therapeutic manipulations of this kinase pathway.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. JNK independent cJun phosphorylation.

Purified B lymphocytes were stimulated through both CD40 and TLR7 for indicated times. Cells were lysed and analyzed by Western blot for A) phospho–cJun, total -cJun and Actin as a loading control and B) CDK4 and cyclin D2. C) Resting splenic B lymphocyets were stimulated through TLR7 and CD40 (R848 1 ug/ml and CD40L) for the indicated times. Cells were lysed and analyzed for phosphorylated MAP kinases by Western blot. D) Mouse high density splenic B cells or E) human peripheral B cells were stimulated through both CD40 and TLR7 for the designated times in the absence or presence of CDK4 inhibitors SU9516 – CDK-I (10 uM) or Fascaplysin (5 uM) for 5 hours. Cells were then lysed and analyzed for phospho-cJun by Western blot. Results are representative of >3 separate experiments.

Figure 2. Effect of CDK inhibition on AP-1 function.

A) 293T cells were transfected with increasing amounts of a plasmid encoding kinase dead CDK4, together with a construct producing murine TLR7 and an AP-1-luciferase reporter plasmid. Empty pRSV.neo plasmid was added to equalize the total amount of transfected DNA. After 24 hours of stimulation with the TLR7 agonist R848, relative amounts of luciferase activity were measured. B) IL-6 production by B cells stimulated with the agonistic anti-mouse CD40 antibody 1C10 and R848 with or without the CDK inhibitor SU9516 was quantified by IL-6-specific ELISA assay, as described in Methods. C) IL-6 production by purified human peripheral B cells stimulated with R848 for 24 hours with or without the CDK inhibitor SU9516 was monitored by ELISA. These data are each representatives of 3 separate experiments.

Figure 3. Effects of CDK4 inhibition on cytokine production and cJun phosphorylation in non-dividing cells.

Differentiated BMDCs were stimulated with the TLR7 agonist R848 in the presence and absence of CDK-I (SU9516) for 6 and 24 hours and monitored for A) cJun phosphorylation and B) IL-6 production, as described in Methods. C) As indicated by CFSE staining, matured BMDCs did not proliferate upon TLR7 stimulation – DCs were either fixed (light grey line) or stimulated with R848 for 48 hours (dark grey line) D) BMDCs from CDK4 deficient mice and CDK4+/+ littermate controls were stimulated with R848 (1 ug/ml) for 24 hours and supernatants subjected to cytokine multiplex analysis. E) BMDCs from CDK4 deficient mice and CDK4+/+ were stimulated with R848 (1 ug/ml) for 6 hours and assayed for cJun phosphorylation. The level of c-Jun phosphorylation in the CDK4 sufficient cells, as determined by the p-c-Jun:actin ratio, was set to 100%. The level of p-c-Jun in CDK4 deficient cells was normalized to the 100%. F) Human monocyte-derived macrophages were stimulated with or without R848 for 6 hours in the presence of medium, DMSO (drug diluent), or the CDK4-specific inhibitor CINK. Cell lysates were then prepared and analyzed by Western blot for the presence of phospho-cJun. Actin was measured as a loading control. G) Human monocyte-derived macrophages were stimulated with or without R848 for 24 hours in the presence of DMSO, or the CDK4-specific inhibitor CINK. Supernatants were collected and analyzed for cytokines using multiplex technology. (*) indicates statistical significance – P-value

Figure 4. CDK4 activity in non-dividing cells.

A) Mixed myeloid cells expanded from bone marrow of C57Bl/6 mice were stained with CFSE prior to stimulation with the TLR7 agonist R848. After 48 and 72 hours cells were fixed and stained for phosphoryated Rb protein on the CDK4 specific site Ser 780. Cells in gate M1 are CFSE high and represent non-dividing cells. The percentage of cells within this gate remained approximately 26% for three days. Cells in gates M2 and M3 are CFSE low and represent cells that have gone through 1 or more than 1 division respectively. From day 2 to day 3 the % of M2 cells has decreased while the percent of cells in M3 has increased indicating ongoing proliferation. B) A histogram of MFI for phospho-Rb from each of the M gates indicates phosphorylation of Rb protein in all stages of division (line represents MFI of non-stimulated cells). C) Histogram of cells from M1 gate stained for phospho-Rb protein after 48 hours of R848 stimulation. Peak 1 is the isotype control of stimulated cells, peak 2 is the p-Rb staining of non-stimulated cells, and peak 3 is the p-Rb staining of R848 stimulated cells. These data are representative of two individual experiments.

Figure 5. Phosphorylation of cJun by cellular CDK4.

A) Purified recombinant CDK4:cyclinD complex was reacted with cJun and GSK GST fusion proteins to determine specificity of phosphorylation by CDK4. Western blotting with phospho-specific Abs was used to monitor the phosphorylation of the GST-fusion proteins. These data are representative of 3 separate experiments. B) CDK4 was immunoprecipitated from differentially stimulated B lymphocytes and reacted with GST-cJun fusion protein as described in Methods. The presence of cyclin D2 and phosphorylation of c-Jun was analyzed by Western Blot using anti-Ser 73 phospho-cJun Abs. Blotting against CDK4 was used as a loading control. Quantitative analysis of chemiluminescent band development enabled comparison of relative protein amounts between treatments (bar graphs). These data are representative of 2 separate experiments.