Elsevier

Blood Cells, Molecules, and Diseases

Volume 39, Issue 3, November–December 2007, Pages 272-277
Blood Cells, Molecules, and Diseases

Mip/LIN-9 can inhibit cell proliferation independent of the pocket proteins

https://doi.org/10.1016/j.bcmd.2007.05.006Get rights and content

Abstract

Progression through the G1-phase of the cell cycle requires that cyclin D and CDK4 phosphorylate pRB and the other pocket proteins, p107 and p130. Cyclin E and CDK2 further phosphorylate pRB to complete its inactivation and allow the cell to enter S-phase. These phosphorylation events lead to the inactivation of the antiproliferative effect of the pocket proteins. The pocket proteins are the main targets of CDK4, and its unregulated activity can contribute to carcinogenesis. Mip/LIN9 is a recently described protein with growth suppressor, as well as growth promoting effects due to its ability to stabilize B-Myb and induce genes required for S phase and mitosis. The finding that a mutation that deletes the first 84 amino acids of Mip/LIN-9 corrects the defects of the CDK4 knockout mouse suggests that it should have a growth repressor effect that is blocked by CDK4. However, overexpression of cyclin D only partially blocks the inhibitory effect of Mip/LIN-9 on cell proliferation. Here, we performed experiments to further understand the antiproliferative effect of Mip/LIN-9 within the context of the pocket proteins. Our results suggest that there is a pocket protein-independent mechanism of the Mip/LIN-9 antiproliferative effect since it can be observed in cells with ablation of the three members of the family, and in NIH3T3 cells expressing the adenovirus E1A-12S protein. Altogether, the independence from the pocket proteins and the partial blockade of the antiproliferative effect produced by expression of cyclin D suggest that the role of Mip/LIN-9 downstream of CDK4 may be more closely related to the activation of B-Myb and the induction of S/M genes. Importantly, the regulatory effect of CDK4 is not due to direct phosphorylation of Mip/LIN-9 by this kinase or even CDK2, suggesting an indirect mechanism such as phosphorylation of the pocket proteins.

Introduction

Quiescent cells are induced to divide following the addition of growth factors. In order for cell division to be autonomous of external stimuli it must overcome a regulatory mechanism termed the restriction point [1], [2], [3]. A key event at this point represents the inactivation of the retinoblastoma gene product, pRB, by phosphorylation thereby freeing the E2F transcription factor to activate the genes necessary for cell cycle progression [4], [5], [6]. This phosphorylation is accomplished by specific cyclin/CDK modules. CyclinD/CDK4 is the primary kinase to phosphorylate pRB in early G1 [7], [8]. Following expression of cyclin E, pRB is further phosphorylated by cyclinE/CDK2 leading to its complete inactivation [9], [10], [11], [12]. The importance of this event is highlighted by activating mutations of the cyclin D/CDK4 machinery commonly found in cancer cells [13], [14], [15]. Furthermore, overexpressing pRB in tissue culture can lead to a G1 cell cycle arrest [16].

The CDK4 knockout mouse has provided insight to the importance of this G1 kinase [17], [18]. The phenotype of the CDK4 knockout mouse was characterized by small body mass, infertility in both sexes and diabetes. Each characteristic could be related to a defect in proliferation. During continuous culture in complete medium CDK4−/− MEFs are completely indistinguishable from wild-type MEFs. However, when they are serum starved to quiescence and then released, the CDK4−/− MEFs have a delay in cell cycle reentry demonstrating the role of CDK4 in initiating the cell cycle [17], [18].

Mip/LIN-9 is the mammalian member of a new family of proteins, which also includes C. elegans LIN-9, Drosophila mip130 and aly, and A. thaliana always early [19], [20], [21], [22]. C. elegans LIN-9 was the first member discovered [19]. It participates in a pRB-like pathway that negatively regulates vulva differentiation and was shown to partially correct a cyclin D mutant in C. elegans [23], [24]. Mip/LIN-9 was first described by Gagrica et al. as a suppressor of oncogenesis in collaboration with pRB [25]. Sandoval et al. described that expression of Mip/LIN-9 inhibits cell proliferation and that this effect is partially overcome by cyclin D [26]. Furthermore, the CDK4−/− phenotype could be corrected by crossing with mice carrying an 84 amino acid N-terminal deletion of Mip/LIN-9, termed Δ84. Remarkably, MEFs generated from this cross had a restored cell cycle reentry following serum-starvation and a normalization of the expression of E2F-regulated genes. These findings demonstrate that the antiproliferative effect of Mip/LIN-9 is negatively regulated by Cyclin D/CDK4.

Interestingly, Mip/LIN-9 is also critical for the progression through S-phase and mitosis through an interaction with the transcription factor B-Myb [27]. In the absence of Mip/LIN-9, B-Myb is likely degraded leading to an impaired expression of S/M-phases genes such as cyclin A, cyclin B and CDK1. Additionally, co-expression of Mip/LIN-9 and B-Myb leads to activation of the same promoters in cells with unregulated CDK4 activity.

We recently have determined that Mip/LIN-9 specifically interacts with p107, p130 and E2F4 but not with pRB or activating E2Fs [28]. Moreover, Mip/LIN-9 has mutually exclusive interaction with p107,p130/E2F4 in G0/G1 and with B-Myb in G1/S phases of the cell cycle [28]. The dissociation of Mip/LIN-9 from p107,p130/E2F4 is likely driven by an increase in CDK4 activity, since it does not re-associate with the repressor complex until the activity of this CDK falls to baseline levels in the next cell cycle. These findings support a model in which CDK4 regulates the expression of S/M genes via Mip/LIN-9 [28].

Importantly, although the mutation of Mip/LIN-9 corrected the CDK4 null phenotype, overexpression of cyclin D resulted in only a partial block of the antiproliferative effect of Mip/LIN-9 in NIH/3T3 cells [26]. This finding supports the concept that Mip/LIN-9 may affect cell proliferation by different mechanisms. In this report, we tested whether Mip/LIN-9 was able to inhibit cell proliferation only within the context of the pocket proteins and the effect of CDK4 on Mip/LIN-9 phosphorylation. Our results demonstrate that overexpression of Mip/LIN-9 in cells that lack pRB, p107 and p130 still inhibits cell proliferation. Moreover, the effect of Mip/LIN-9 is not abrogated by E1A-12S, which encodes a protein capable of blocking the growth inhibitory effect of all pocket proteins, further supporting a pocket protein-independent component in the antiproliferative effect of Mip/LIN-9. Surprisingly, the 13S form of E1A was able to inhibit the growth inhibitory effect of Mip/LIN-9 indicating that the zinc finger region, present only in the 13S form, is responsible for overcoming the antiproliferative effect of Mip/LIN-9 [29]. Finally, our results also suggest that the regulatory effect of CDK4 is not exerted via direct phosphorylation of Mip/LIN-9. Altogether, these data supports the concept that the growth inhibitory effect of Mip/LIN-9 has a component that is independent of cyclin D/CDK4 and the pocket proteins, and that the role of this G1 kinase may be more closely related to the release of Mip/LIN-9 from the p107,p130/E2F4 repressor complex to allow the association with B-Myb and the induction of S/M genes.

Section snippets

Cell culture

All cell lines were cultured in DMEM supplemented with 10% FBS. pLXSN retrovirus was produced using the BOSC23 packaging cell line or PA317.

Colony formation

Cell lines were plated in 100 mm plates and infected the next day with pLXSN, pLXSN-Mip/LIN-9L, or pLXSN-Mip/LIN-9S. Following two days of infection the medium was changed to selection medium containing 500 μg/mL G418. Selection continued for up to two weeks with the medium changed every 3 days. Following selection plates were fixed with methanol, stained

Results

Previous reports indicated that Mip/LIN-9 has a negative effect on cell growth [25], [26]. To further assess the effect of Mip/LIN-9 on cell proliferation, we performed colony assays on a broad range of cell lines. A retroviral vector, pLXSN, containing either the full length Mip/LIN-9-L (MW approximately 62,000) or the splice variant Mip/LIN-9-S (MW approximately 54,000) or empty vector was used to infect a variety of cell lines. Following selection for ten to fourteen days, the colonies were

Discussion

Several lines of evidence place Mip/LIN-9 downstream of CDK4. First, the CDK4−/− mouse has proliferative defects that are corrected by crossing with Δ84 Mip/LIN-9 animals, [17], [18], [26]. Second, overexpression of Mip/LIN-9 in NIH/3T3 causes a cell cycle arrest and this is partially overcome by coexpressing cyclin D [26]. In this paper, we explored the relationship between Mip/LIN-9 and the best-known substrates of CDK4, the pocket protein, as well as the relationship between CDK4 activity

Acknowledgments

We thank Drs. M. Classon and T. Jacks for the pRB and TKO cells. We also appreciate Dr. J. Cook for the NIH/3T3 cells expressing different forms of the adenovirus E1A protein.

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    This work has been supported by The National Institutes of Health Grant GM54709 (ORC). MP was supported by an NRSA/NIH Institutional T32 training grant, “Training Program in Signal Transduction and Cellular Endocrinology”, T32 DK07739 from the NIDDK. This paper is based on a presentation at a Focused Workshop of the 4th Myb Workshop entitled: “Mip/LIN-9 links negative and positive regulators of the cell cycle” sponsored by The Leukemia & Lymphoma Society held in Civitella Alfedena, May 20–24th, 2007.

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