Biochemical Characterization of p16 - and p18-containing Complexes in Human Cell Lines

Abstract

The regulation of the D-type cyclin-dependent kinase (CDK4 and CDK6) activity appears to be the key step in the progression of eukaryotic cells through the G 1 cell cycle phase. One of the mechanisms involved in this process is the binding of some small proteic inhibitors, with a molecular mass ranging between 14 and 20 kDa, to these CDKs. We have evaluated the amount of two such inhibitors, namely p16INK4 and p18, in normal and transformed cells, as well as the biochemical features of the macromolecular complexes containing these proteins. The results obtained indicated that (i) p18 gene expression, unlike p16 INK4 gene, is not regulated by pRb status, (ii) no evident relationship exists between the expression of p16 INK4 and p18 genes, (iii) significant amounts of the two proteins are not bound to CDKs but occur as free molecules, (iv) each inhibitor forms a complex with the CDK protein with a 1:1 stoichiometry, and (v) a competition exists between cyclin D and the inhibitor protein toward the CDK protein resulting in the absence of detectable cellular free kinase. Moreover, employing the human native partially purified p16 INK4or the pure recombinant protein, we have been able to demonstrate in vitro the dissociation of CDK4-cyclin D1 complex and the formation of CDK4-p16 INK4 bimolecular complex. Our findings suggest that during the cell division cycle the members of the p16 INK4 protein family and cyclin Ds compete for binding to CDK4/CDK6 and that their quantitative ratio is essential for G 1 3 S transition.In eukaryotes the progression through the cell cycle is due to a biochemical cycle in which distinct cyclin-dependent serinethreonine kinases (CDKs) 1 are sequentially activated by different cyclins (1, 2). Then activated CDKs regulate their target molecules by phosphorylation. Finally, these downstream molecules carry out the steps that ultimately allow the ordered development of the cell division cycle (3-7). Thus, the regulation of CDK activity is the key event in the cell cycle progression.The level of the various CDK proteins generally does not vary remarkably during the cycle, whereas the amount of each cyclin undergoes dramatic changes. During the early G 1 phase, in particular, there occurs the accumulation of three D-type cyclins (D1, D2, and D3) (8 -11), which assemble into holoenzymes with either CDK4 (5,7,12) or, in a minor number of cell types, CDK6 (13,14). Interaction between cyclin Ds and these CDKs is not sufficient to activate the kinase activity of the holoenzyme, since functioning CDKs also require phosphorylation at a single threonyl residue (15). Additional proteins are normal constituents of complexes formed between D-type cyclins and CDK4 (or CDK6), including the proliferating cell nuclear antigen and a small protein named p21. Proliferating cell nuclear antigen, the processivity factor required by eukaryotic DNA polymerases ␦ and ⑀ (16 -21), is involved in the control of the rate of chromosome replication as well as in the repair of damaged DNA (22,23). p21 is a negative regul...

p27Kip1 accumulation is associated with retinoic-induced neuroblastoma differentiation: evidence of a decreased proteasome-dependent degradation

“…To evaluate this hypothesis, we exploited a method based on a gel-®ltration chromatographic separation of cell extracts followed by immunoblotting analysis of each fraction (Della Ragione et al, 1996). The technique employs a Superdex-75 HR column, which provides a very high resolution in a molecular mass range lower than 80 kDa.…”

INK4 cell cycle inhibitors direct transcriptional inactivation of NF-κB

“…The ankyrin repeats in the INK4 proteins (Luh et al, 1997), which represent a known protein-protein interaction motif (Bork, 1993), are also found to be important in the IkB family of NF-kB inhibitors (Hatada et al, 1992(Hatada et al, , 1993Wulczyn et al, 1992, andreviewed in Wulczyn et al, 1996). Signi®cant amounts of INK4 molecules occur as free molecules in human cell lines (Della Ragione et al, 1996), thus it is possible that they have other targets than the formally known Cdk4/Cdk6. Suggestions about their cellular role is given by Chan et al (1995) who showed that p19INK4 interacts with the apoptosis inducing orphan steroid receptor Nur77.…”

“…The ankyrin motif in INK4 proteins, a feature of peptides involved in protein-protein interaction (Bork, 1993), and the fact that signi®cant amounts of cellular p16INK4 and p18INK4 occur in a free form (Della Ragione et al, 1996) suggest additional roles of INK4 proteins in cell physiology. In this scenario, it was shown that p19INK4 interacts with Nur77 (Chan et al, 1995) and yeast Pho81 (containing high similarity to mammalian p16INK4) interacts with Pho4 (Hirst et al, 1994), proteins which are totally unrelated to Cdks.…”

“…As a prerequisite for e cient interaction of endogenous INK4 molecules with NF-kBp65 the cells have to respond to mitogen stimulation (meaning NF-kB inhibitor degradation) and must display unbound INK4 molecules. Transformed cells as well as normal cells could have high abundance of INK4 molecules (Della Ragione et al, 1996) suggesting that in such cells mitogen induced NF-kB activation could be a ected by INK4 molecules.…”

Functional evaluation of tumour-specific variants of p16INK4a/CDKN2A: correlation with protein structure information

“…A second point is that any assay conducted in vitro, such as CDK binding or kinase inhibition, may not be a true re¯ection of the in vivo situation. For example, it is now clear that cyclin D-dependent kinase activity is associated with high molecular weight complexes in vivo and that D cyclins are rarely if ever found in simple binary associations with CDK4 or CDK6 (Della Ragione et al, 1996;Mahony et al, 1998;Musgrove et al, 1998;McConnell et al, 1999). Similarly, although p16 INK4a does participate in binary complexes with CDKs, it does not associate detectably with D cyclins in vivo (Hall et al, 1995;Parry et al, 1995;Guan et al, 1996;McConnell et al, 1999).…”