Conformation-dependent phosphorylation of p53

Abstract

Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions. Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 ((His)p53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and beta subunit of casein kinase II (CKIIbeta) as well as by kinases present in whole cell extracts (WCEs). We demonstrate that PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53. We further demonstrate JNK interaction with GST-p53 and the ability of JNK to phosphorylate truncated forms of GST-p53 or full-length (His)p53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53wt) undergoes better phosphorylation by CKIIbeta and by WCE kinases than mutant forms of p53 at amino acid 249 (p53(249)) or 273 (p53(273)). Moreover, shifting the kinase reaction's temperature from 37 degrees C to 18 degrees C reduces the phosphorylation of mutant p53 to a greater extent than of p53wt. Comparing truncated forms of p53 revealed that the ability of CKIIbeta, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97-155 within the DNA-binding domain region. Among three 20-aa peptides spanning this region we have identified residues 97-117 that increase p53 phosphorylation by CKIIbeta while inhibiting p53 phosphorylation by PKA or WCE kinases. The importance of this region is further supported by computer modeling studies, which demonstrated that mutant p53(249) exhibits significant changes to the conformation of p53 within amino acids 97-117. In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, as for the phosphorylation pattern generated by the same kinase.

Figure 1

(A) Effect of WCE kinases on phosphorylation of p53. GST-p53 wild-type (amino acids 1–393; molecular mass 80 kDa) or truncated forms (as indicated by arrows) were incubated with WCE as source of kinases. The solid-phase kinase reaction initiated by addition of [γ-³²P]ATP for 15 min at room temperature, followed by washes of glutathione beads-bound GST-p53 and elution of p53 from beads by SDS-loading buffer. Samples were separated on SDS/15% PAGE and autoradiographed. Molecular masses are indicated at left in kDa. GST-c-jun^5–89 used as control is shown in the left lane. (B) CKII phosphorylation of p53. Solid-phase kinase assay with respective GST-p53 proteins (position of major phosphorylation site indicated by arrow) was performed in the presence of 40 ng of the catalytic subunit of CKIIβ. (C) PKA phosphorylation of p53. GST-p53 proteins used for solid-phase kinase reaction using catalytic subunit of PKA (80 ng) as source of kinase. Position of major phosphorylation site is indicated by arrows. (D) Mapping of PKA phosphorylation sites on p53. Products of solid-phase kinase reaction using PKA (2 μg of enzyme per reaction) or WCE (2 μg) were washed and then separated on SDS/PAGE. Pattern of endogenous p53 phosphorylation is shown in IP lane, in which cells were labeled with [³²P]-orthophosphate (100 μCi) for 4 h before p53 was immunoprecipitated (200 ng of p53 antibody per 1 mg of protein). Bands corresponding to each of the GST-p53 proteins were excised and subjected to trypsinization followed by separation on an isoelectric focusing gel (pH range 3.5–10). Peptides were analyzed by autoradiography.

Figure 2

(A) JNK phosphorylation of GST-p53 proteins. Full-length or truncated forms of GST-p53 proteins were used for solid-phase kinase reaction using JNK purified from WCE. Respective bands were excised, subjected to digestion with trypsin, and analyzed further by separation on isoelectric focusing gel. (B) p53 phosphorylation by JNK. Solid-phase kinase reactions were performed using bacterially produced active JNK (200 ng) or PKA (2 μg) as the source of kinase and GST p53^1–393 (lanes 2, 3), thrombin-digested p53 that lacks the GST portion (lanes 4, 5), _Hisp53 (lane 1), or GST c-jun^5–89 (lanes 1–3) as substrates. (C) CKIIβ, PKA, and JNK phosphorylation of _Hisp53 and GST-p53. GST-p53 or _Hisp53 substrates were used in solid-phase kinase reaction using CKIIβ (40 ng), PKA (80 ng), or JNK (200 ng) at 18° or 37°C. Arrows point to the position of the GST or histidine-tagged protein. (D) JNK binding to p53. GST-p53 constructs were incubated with protein extracts for either 10 or 30 min at room temperature before beads-bound material was washed and analyzed on Western blots using antibodies to JNK. IP lane represents proteins immunoprecipitated with antibodies to p53 and analyzed by Western blotting with antibodies to JNK. Molecular mass (kDa)is shown at the right, and molecular masses of proteins recognized by JNK antibodies are indicated on the left.

Figure 3

(A) Effect of temperature on phosphorylation of wild-type and mutant forms of p53. GST-p53 was subjected to solid-phase kinase reaction using the kinases at 18°C or 37°C. (B) Mapping of mutant p53 phosphorylation. Products of solid-phase p53 phosphorylation at indicated temperatures were separated on SDS/PAGE, and p53 bands, identified through autoradiography, were cut, excised, and digested with trypsin before separation on an isoelectricfocusing gel. Resulting phosphorylated peptides are indicated by the arrowheads on the sides. Phosphorylated peptides identified in wild-type, but not mutant, forms of p53 are indicated by arrowheads on lane 3 (compare with lanes 4 and 5).

Figure 4

(A) Effect of DNA-binding domain-derived peptides on p53 phosphorylation. GST-p53^1–393 was subjected to solid-phase kinase reaction with WCE, in the presence of peptides 7–9 before addition of [γ³²P]ATP (A lanes), before and during kinase reaction (B lanes), or only with the addition of [γ³²P]ATP (C). Lower bands represent staining of the GST-p53 to indicate equal loading. (B) Effect of peptides on phosphorylation of p53 by PKA and WCE. GST-p53^1–393 was subjected to solid-phase phosphorylation (i.e., washes performed before addition of ATP; lanes C and 7–9) or with [γ³²P]ATP added without intermediate washes (lanes A and 7*) by PKA or WCE, respectively, in the presence of indicated peptides. (C) Effect of P7 on p53 phosphorylation by CKIIβ, PKA, and WCE. GST-p53 subjected to solid-phase kinase reaction in the presence of P7 and kinases.

Figure 5

Stereoview of the C^α trace of the segment 96–155 of two p53 proteins: wild-type (yellow) and Arg²⁴⁹ → Trp mutant (red). The phosphorylation domain from residues 96–117 is shown in a tubular form for both proteins.