Martin F. Crook

A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression

The cyclin‐dependent kinase inhibitor, p27Kip1, which regulates cell cycle progression, is controlled by its subcellular localization and subsequent degradation. p27Kip1 is phosphorylated on serine 10 (S10) and threonine 187 (T187). Although the role of T187 and its phosphorylation by Cdks is well‐known, the kinase that phosphorylates S10 and its effect on cell proliferation has not been defined. Here, we identify the kinase responsible for S10 phosphorylation as human kinase interacting stathmin (hKIS) and show that it regulates cell cycle progression. hKIS is a nuclear protein that binds the C‐terminal domain of p27Kip1 and phosphorylates it on S10 in vitro and in vivo, promoting its nuclear export to the cytoplasm. hKIS is activated by mitogens during G0/G1, and expression of hKIS overcomes growth arrest induced by p27Kip1. Depletion of KIS using small interfering RNA (siRNA) inhibits S10 phosphorylation and enhances growth arrest. p27−/− cells treated with KIS siRNA grow and progress to S/G2similar to control treated cells, implicating p27Kip1 as the critical target for KIS. Through phosphorylation of p27Kip1 on S10, hKIS regulates cell cycle progression in response to mitogens.

Bone marrow–derived immune cells regulate vascular disease through a p27 Kip1 -dependent mechanism

The cyclin-dependent kinase inhibitors are key regulators of cell cycle progression. Although implicated in carcinogenesis, they inhibit the proliferation of a variety of normal cell types, and their role in diverse human diseases is not fully understood. Here, we report that p27(Kip1) plays a major role in cardiovascular disease through its effects on the proliferation of bone marrow-derived (BM-derived) immune cells that migrate into vascular lesions. Lesion formation after mechanical arterial injury was markedly increased in mice with homozygous deletion of p27(Kip1), characterized by prominent vascular infiltration by immune and inflammatory cells. Vascular occlusion was substantially increased when BM-derived cells from p27(-/-) mice repopulated vascular lesions induced by mechanical injury in p27(+/+) recipients, in contrast to p27(+/+) BM donors. To determine the contribution of immune cells to vascular injury, transplantation was performed with BM derived from RAG(-/-) and RAG(+/+) mice. RAG(+/+) BM markedly exacerbated vascular proliferative lesions compared with what was found in RAG(-/-) donors. Taken together, these findings suggest that vascular repair and regeneration is regulated by the proliferation of BM-derived hematopoietic and nonhematopoietic cells through a p27(Kip1)-dependent mechanism and that immune cells largely mediate these effects.

Disruption of Protein Arginine N-Methyltransferase 2 Regulates Leptin Signaling and Produces Leanness In Vivo Through Loss of STAT3 Methylation

Rationale: Arginine methylation by protein N-arginine methyltransferases (PRMTs) is an important posttranslational modification in the regulation of protein signaling. PRMT2 contains a highly conserved catalytic Ado-Met binding domain, but the enzymatic function of PRMT2 with respect to methylation is unknown. The JAK-STAT pathway is proposed to be regulated through direct arginine methylation of STAT transcription factors, and STAT3 signaling is known to be required for leptin regulation of energy balance. Objective: To identify the potential role of STAT3 arginine methylation by PRMT2 in the regulation of leptin signaling and energy homeostasis. Methods and Results: We identified that PRMT2−/− mice are hypophagic, lean, and have significantly reduced serum leptin levels. This lean phenotype is accompanied by resistance to food-dependent obesity and an increased sensitivity to exogenous leptin administration. PRMT2 colocalizes with STAT3 in hypothalamic nuclei, where it binds and methylates STAT3 through its Ado-Met binding domain. In vitro studies further clarified that the Ado-Met binding domain of PRMT2 induces STAT3 methylation at the Arg31 residue. Absence of PRMT2 results in decreased methylation and prolonged tyrosine phosphorylation of hypothalamic STAT3, which was associated with increased expression of hypothalamic proopiomelanocortin following leptin stimulation. Conclusions: These data elucidate a molecular pathway that directly links arginine methylation of STAT3 by PRMT2 to the regulation of leptin signaling, suggesting a potential role for PRMT2 antagonism in the treatment of obesity and obesity-related syndromes.

KIS protects against adverse vascular remodeling by opposing stathmin-mediated VSMC migration in mice

Vascular proliferative diseases are characterized by VSMC proliferation and migration. Kinase interacting with stathmin (KIS) targets 2 key regulators of cell proliferation and migration, the cyclin-dependent kinase inhibitor p27Kip1 and the microtubule-destabilizing protein stathmin. Phosphorylation of p27Kip1 by KIS leads to cell-cycle progression, whereas the target sequence and the physiological relevance of KIS-mediated stathmin phosphorylation in VSMCs are unknown. Here we demonstrated that vascular wound repair in KIS-/- mice resulted in accelerated formation of neointima, which is composed predominantly of VSMCs. Deletion of KIS increased VSMC migratory activity and cytoplasmic tubulin destabilizing activity, but abolished VSMC proliferation through the delayed nuclear export and degradation of p27Kip1. This promigratory phenotype resulted from increased stathmin protein levels, caused by a lack of KIS-mediated stathmin phosphorylation at serine 38 and diminished stathmin protein degradation. Downregulation of stathmin in KIS-/- VSMCs fully restored the phenotype, and stathmin-deficient mice demonstrated reduced lesion formation in response to vascular injury. These data suggest that KIS protects against excessive neointima formation by opposing stathmin-mediated VSMC migration and that VSMC migration represents a major mechanism of vascular wound repair, constituting a relevant target and mechanism for therapeutic interventions.

The cell cycle regulator p27Kip1 interacts with MCM7, a DNA replication licensing factor, to inhibit initiation of DNA replication.

The G1/S phase restriction point is a critical checkpoint that interfaces between the cell cycle regulatory machinery and DNA replicator proteins. Here, we report a novel function for the cyclin‐dependent kinase inhibitor p27Kip1 in inhibiting DNA replication through its interaction with MCM7, a DNA replication protein that is essential for initiation of DNA replication and maintenance of genomic integrity. We find that p27Kip1 binds the conserved minichromosome maintenance (MCM) domain of MCM7. The proteins interact endogenously in vivo in a growth factor‐dependent manner, such that the carboxyl terminal domain of p27Kip1 inhibits DNA replication independent of its function as a cyclin‐dependent kinase inhibitor. This novel function of p27Kip1 may prevent inappropriate initiation of DNA replication prior to S phase.

GA-binding protein regulates KIS gene expression, cell migration, and cell cycle progression

The cyclin‐dependent kinase inhibitor p27Kip1 arrests cell cycle progression through G1/S phases and is regulated by phosphorylation of serine/ threonine residues. Recently, we identified the serine/ threonine kinase, KIS, which phosphorylates p27Kip1 on serine 10 leading to nuclear export of p27Kip1 and protein degradation. However, the molecular mechanisms of transcriptional activation of the human KIS gene and its biological activity are not known. We mapped the transcription initiation site ~116 bp 5′ to the translation start site, and sequences extending to ‐141 were sufficient for maximal promoter activity. Mutation in either of two Ets‐binding sites in this region resulted in an approximately 75–80% decrease in promoter activity. These sites form at least 3 specific complexes, which contained GA‐binding protein (GABP). Knocking down GABPα by siRNA in vascular smooth muscle cells (VSMCs) diminished KIS gene expression and reduced cell migration. Correspondingly, in serum stimulated GABPα‐deficient mouse embryonic fibroblasts (MEFs), KIS gene expression was also significantly reduced, which was associated with an increase in p27Kip1 protein levels and a decreased percentage of cells in S‐phase. Consistent with these findings, following vascular injury in vivo, GABPα‐heterozygous mice demonstrated reduced KIS gene expression within arterial lesions and these lesions were significantly smaller compared to GABP+/+ mice. In summary, serum‐responsive GABP binding to Ets‐binding sites activates the KIS promoter, leading to KIS gene expression, cell migration, and cell cycle progression.— Crook M. F., Olive, M., Xue, H.‐H., Lange‐nickel T. H., Boehm, M., Leonard, W. J., Nabel E. G. GA‐binding protein regulates KIS gene expression, cell migration, and cell cycle progression. FASEB J. 22, 225–235 (2008)

Discovery of benzimidazole oxazolidinediones as novel and selective nonsteroidal mineralocorticoid receptor antagonists.

Elaboration of the oxazolidinedione series led to replacement of the exocyclic amides with substituted benzimidazoles. The structure-activity relationship (SAR) exploration resulted in the discovery of potent and selective nonsteroidal mineralocorticoid receptor (MR) antagonists with significantly improved microsomal stability and pharmacokinetic (PK) profile relative to the HTS hit 1a. One compound 2p possessed comparable efficacy as spironolactone (SPL) at 100 mg/kg (p.o.) in the rat natriuresis model. As such, this series was validated as a lead series for further optimization.