Regulation of cell division by intrinsically unstructured proteins: intrinsic flexibility, modularity, and signaling conduits - PubMed (original) (raw)
Review
Regulation of cell division by intrinsically unstructured proteins: intrinsic flexibility, modularity, and signaling conduits
Charles A Galea et al. Biochemistry. 2008.
Abstract
It is now widely recognized that intrinsically unstructured (or disordered) proteins (IUPs or IDPs) are found in organisms from all kingdoms of life. In eukaryotes, IUPs are highly abundant and perform a wide range of biological functions, including regulation and signaling. Despite an increased level of interest in understanding the structural biology of IUPs and IDPs, questions regarding the mechanisms through which disordered proteins perform their biological function(s) remain. In other words, what are the relationships between disorder and function for IUPs? There are several excellent reviews that discuss the structural properties of IUPs and IDPs since 2005 [Receveur-Brechot, V., et al. (2006) Proteins 62, 24-45; Mittag, T., and Forman-Kay, J. D. (2007) Curr. Opin. Struct. Biol. 17, 3-14; Dyson, H. J., and Wright, P. E. (2005) Nat. Rev. Mol. Cell Biol. 6, 197-208]. Here, we briefly review general concepts pertaining to IUPs and then discuss our structural, biophysical, and biochemical studies of two IUPs, p21 and p27, which regulate the mammalian cell division cycle by inhibiting cyclin-dependent kinases (Cdks). Some segments of these two proteins are partially folded in isolation, and they fold further upon binding their biological targets. Interestingly, some portions of p27 remain flexible after binding to and inhibiting the Cdk2-cyclin A complex. This residual flexibility allows otherwise buried tyrosine residues within p27 to be phosphorylated by non-receptor tyrosine kinases (NRTKs). Tyrosine phosphorylation relieves kinase inhibition, triggering Cdk2-mediated phosphorylation of a threonine residue within the flexible C-terminus of p27. This, in turn, marks p27 for ubiquitination and proteasomal degradation, unleashing full Cdk2 activity which drives cell cycle progression. p27, thus, constitutes a conduit for transmission of proliferative signals via post-translational modifications. The term "conduit" is used here to connote a means of transmission of molecular signals which, in the case of p27, correspond to tyrosine and threonine phosphorylation, ubiquitination, and, ultimately, proteolytic degradation. Transmission of these multiple signals is enabled by the inherent flexibility of p27 which persists even after tight binding to the Cdk2-cyclin A complex. Importantly, activation of the p27 signaling conduit by oncogenic NRTKs contributes to tumorigenesis in some human cancers, including chronic myelogenous leukemia (CML) [Grimmler, M., et al. (2007) Cell 128, 269-280] and breast cancer [Chu, I., et al. (2007) Cell 128, 281-294]. Other IUPs may participate in conceptually similar molecular signaling conduits, and dysregulation of these putative conduits may contribute to other human diseases. Detailed study of these IUPs, both alone and within functional complexes, is required to test these hypotheses and to more fully understand the relationships between protein disorder and biological function.
Figures
Figure 1. Regulation of the eukaryotic cell division cycle
(A) Illustration of the various stages of the cell division cycle and the Cdk/cyclin complexes that play roles in regulating progression through the different stages are indicated. Initiation of cell division in G1 phase requires the activity of Cdk4/cyclin D and Cdk6/cyclin D and progression into S phase (when DNA synthesis or replication occurs, “S”) requires Cdk2/cyclin E and Cdk2/cyclin A (and similar complexes with Cdk1). The activity of Cdk1/cyclin B and Cdk1/cyclin A are required for entry into mitosis (“M”). While initially thought to be universal inhibitors of these Cdk/cyclin complexes, p21 and p27 have been show to activate Cdk4/cyclin D and Cdk6/cyclin D under certain circumstances (indicated by arrow). (B) Alignment of sequences of the kinase inhibitory domains (KID) of p27, p21 and p57. The boundaries of sub-domains D1 (blue), LH (black), D2 (red) and 310 (green) are indicated, as is the “RxL” motif which is recognized by cyclin A. (C) Illustration of the domain structure of p21, p27, and p57. PCNA, PCNA binding domain; NLS, nuclear localization signal; QT, QT domain which contains one or more QT motifs that are either known or putative phosphorylation sites. PAPA, domain with multiple repeats of PAPA motif. The locations of known phosphorylation sites, Y74, Y88 and T187 in p27 and T310 in p57, are also indicated.
Figure 2. Structure of p27-KID bound to Cdk2/cyclin A
Crystal structure of p27-KID/Cdk2/cyclin A determined in 1996 by Pavletich and co-workers. The sub-domains of p27, D1, LH, D2 and 310 are indicated using the color scheme of Fig. 1.
Figure 3. Disorder predictions for p27
Three disorder prediction programs were used to analyze the structure of p27, including FoldIndex (A), IUPred (B) and PONDR (C). The approximate locations of sub-domains D1, LH, D2 and 310 are indicated at the top, as is the QT domain.
Figure 4. NMR results on the solution structure and dynamics of isolated p27-KID in solution
(A) Secondary 13Cα chemical shift values (δΔ 13Cα) and (B) {1H}-15N heteronuclear NOE (hetNOE) values for p27-KID. (C-F) Patterns of 1HN-1HN NOEs which indicated the existence of partial secondary structure (as indicated) within sub-domains LH (C), D2 (D and E), and 310 (F) of p27-KID. A and B are taken from Lacy, et al., 2004 (29) and C-F are taken from Sivakolundu, et al., 2005 (30).
Figure 5. The sub-domains of p27 sequentially fold and bind Cdk2/cyclin A
Results from NMR, ITC and SPR support a scheme in which the “RxL” motif within sub-domain D1 of p27 binds first to cyclin A (“1”), followed by folding and docking of sub-domain LH, followed by binding of sub-domain D2 to Cdk2 (with extensive remodeling of Cdk2, followed finally by binding of sub-domain 310 in the ATP binding pocket of Cdk2 (“2”). This sequential scheme provides a mechanism for specificity toward Cdk/cyclin complexes which preserve the binding site for the “RxL” motif within sub-domain D1 of p27. A movie depicting this sequential binding scheme is available at
http://www.stjuderesearch.org/data/kriwackilab/p27.html
. Taken from Lacy, et al., 2004 (29).
Figure 6. Phosphorylation of p27 on Y88 (pY88) ejects only the 310-helix (sub-domain 310) from the ATP binding pocket of Cdk2; other sub-domains of p27 remain bound to Cdk2/cyclin A
(A) NMR chemical shift differences (Δδ) between (A) free p27-KID and p27-KID bound to cyclin A/Cdk2 are compared with those for (B) free pY88-p27-KID and pY88-p27-KID bound to cyclin A/Cdk2. The locations of sub-domains within p27 are shown at the top, as is the amino acid sequence of p27-KID. The asterisk indicated the location of Y88. Amino acid residues are indicated using the single letter code. Note: For technical reasons, a mutant form of p27-KID, with Y89 mutated to F, was used in these experiments. Taken from Grimmler, et al., 2007 (43).
Figure 7. The p27 molecule is a signaling conduit
(A) A single snap-shot from a 13 ns molecular dynamics trajectory illustrating the structure of p27 bound to Cdk2/cyclin A (cyan and magenta, respectively). The 100 residue-long C-terminal domain of p27 (yellow tube), which contains T187 (orange), is intrinsically unstructured and highly dynamic in this trajectory. Also illustrated are two critical tyrosine residues, Y74 and Y88 (red and green, respectively), which are phosphorylated by non-receptor tyrosine kinases (NRTKs). Phosphorylation of Y88 (“Step 1”), and possibly Y74, ejects sub-domain 310 from the ATP binding pocket of Cdk2 (indicate by white arrow), allowing T187 within the flexible C-terminal domain to encounter the Cdk2 active site (“Step 2”, indicated by grey arrow) and be phosphorylated by Cdk2. (B) Scheme illustrating the two-step p27 phosphorylation mechanism involving Y88 and T187 which triggers p27 poly-ubiquitination and 26S proteasomal degradation in both normal and cancer cells. The pseudo-uni-molecular nature of step 2 is illustrated.
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