Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity

Abstract

Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fraction of the human proteome play important roles in numerous cellular functions. ProstateAssociated Gene 4 (PAGE4) is an IDP that acts as a potentiator of the Activator Protein-1 (AP-1) transcription factor. HomeodomainInteracting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51, but only T51 is critical for its activity. Here, we identify a second kinase, CDC-Like Kinase 2 (CLK2), which acts on PAGE4 and hyperphosphorylates it at multiple S/T residues, including S9 and T51. We demonstrate that HIPK1 is expressed in both androgendependent and androgen-independent prostate cancer (PCa) cells, whereas CLK2 and PAGE4 are expressed only in androgendependent cells. Cell-based studies indicate that PAGE4 interaction with the two kinases leads to opposing functions. HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiates c-Jun, whereas CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuates c-Jun activity. Consistent with the cellular data, biophysical measurements (small-angle X-ray scattering, single-molecule fluorescence resonance energy transfer, and NMR) indicate that HIPK1-PAGE4 exhibits a relatively compact conformational ensemble that binds AP-1, whereas CLK2-PAGE4 is more expanded and resembles a random coil with diminished affinity for AP-1. Taken together, the results suggest that the phosphorylation-induced conformational dynamics of PAGE4 may play a role in modulating changes between PCa cell phenotypes. A mathematical model based on our experimental data demonstrates how differential phosphorylation of PAGE4 can lead to transitions between androgen-dependent and androgen-independent phenotypes by altering the AP-1/androgen receptor regulatory circuit in PCa cells.intrinsic disorder | androgen resistance | prostate cancer | PAGE-4 | phenotypic heterogeneity C ontrary to conventional wisdom that structure defines protein function (1), it is now increasingly evident that a large fraction of the human proteome is composed of intrinsically disordered proteins (IDPs) lacking rigid 3D structure (2-4). IDPs exist as conformational ensembles that are highly malleable, facilitating their interactions with multiple partners. These interactions are "wired" to form scale-free networks (5, 6) that represent the main conduit of information flow in the cell. Furthermore, the organization and properties of such protein interaction networks are evolutionarily conserved, underscoring their functional significance (7).By occupying hub positions in such networks, IDPs play critical roles in many biological processes, such as transcription, translation, and signaling (8, 9). IDPs also participate in higher order phenomena, such as regulation of the cell division cycle (10-12), circadian rhythmicity (13, 14), and phenotypic plasticity (15, 16). Recent evidence suggests that several IDPs can act in a prion-like manner to create protein-based molecular memories that drive the emergence and inheritance of biological traits (17), furt...

Structural metamorphism and polymorphism in proteins on the brink of thermodynamic stability

“…Thus, multi-site phosphorylation can promote structure 23 or disrupt it. 111 Similar types of transient long-range interactions have also been observed in other flexible polypeptide chains where they can mask or attenuate the function of a ligand binding site. For example, the high affinity Bin1-SH3 binding site of Myc (1-88) consists of an approximately 12-residue motif which, when incorporated into an isolated short peptide, binds to Bin1-SH3 with a K D of 4.2 μM.…”

“…The large differences in the ensembles are reflected in opposing functions – HIPK1‐PAGE4 binds the transcription factor Jun/Fos more tightly and potentiates c‐Jun, whereas CLK2‐PAGE4 attenuates c‐Jun activity. Thus, multi‐site phosphorylation can promote structure or disrupt it …”

“…In prostate cancer cells, PAGE4 is differentially phosphorylated by two kinases, Homeodomain Interacting Protein Kinase 1 (HIPK1) and CDC-Like Kinase 2 (CLK2). [110][111][112] Despite both phosphorylated forms being disordered and flexible, biophysical measurements show that HIPK1-PAGE4 has a less disordered conformational ensemble than CLK2-PAGE4, primarily due to the presence of acidic and basic motifs that form transient but stabilizing electrostatic interactions. These long-range effects are disrupted when PAGE4 is hyper-phosphorylated by CLK2 because phosphorylation occurs at multiple sites in or near the basic motifs, effectively neutralizing the transient electrostatic interactions and leading to a more extended ensemble of conformers (Fig.…”

“…Proteins on the brink of stability can undergo a continuum of order/disorder transitions. (A) Examples of transitions from top left to bottom right: Transition between the extended and collapsed disordered states of prostate associated Gene 4 (PAGE4), modulated by phosphorylation; disorder‐to‐order transition of 4E‐BP2 induced by phosphorylation; order‐to‐order fold switching between G A98 and G B98 , triggered by single amino acid changes or ligand binding . In contrast, stable proteins such as subtilisin (shown in dark blue) do not undergo such changes.…”

“…Indeed, while IDPs have different sequence characteristics from metamorphic proteins, it is interesting to note that many IDPs are close to the boundary between disorder and order in plots of mean net charge versus mean hydrophobicity. 14 Thus, the examples of fold switching by marginally stable proteins and disorder/order transitions in marginally unstable IDPs are conceptually similar, representing different 111 disorder-to-order transition of 4E-BP2 induced by phosphorylation; 23 order-to-order fold switching between G A98 and G B98 , triggered by single amino acid changes or ligand binding. 64 points on the continuum of order/disorder transitions.…”