Cloning and Characterization of a Novel Protein Kinase That Impairs Osteoblast Differentiation in Vitro

Abstract

The bone morphogenic proteins (BMPs) play a key role in skeletal development and patterning. Using the technique of differential display polymerase chain reaction (ddPCR), we have identified a novel gene whose expression is increased during BMP-2-induced differentiation of the prechondroblastic cell line, MLB13MYC clone 17, to an osteoblastic phenotype. The 6.5-kilobase mRNA recognized by this ddPCR product is increased 10-fold by BMP-2 treatment of the MLB13MYC clone 17 cells. The mRNA recognized by this ddPCR product is also increased as MC3T3-E1 cells recapitulate the program of osteoblast differentiation during prolonged culture. The full-length transcript corresponding to this ddPCR product was cloned from a MLB13MYC clone 17 cell cDNA library. Analysis of the deduced amino acid sequence demonstrated that this gene encodes a novel 126-kDa putative serine/threonine protein kinase containing a nuclear localization signal. The kinase domain, expressed in Escherichia coli, is capable of autophosphorylation as well as phosphorylation of myelin basic protein. The gene was, therefore, named BIKe (BMP-2-Inducible Kinase). The BIKe nuclear localization signal is able to direct green fluorescent protein to the nucleus in transfected COS-7 cells. When stably expressed in MC3T3-E1 cells, BIKe significantly decreases alkaline phosphatase activity and osteocalcin mRNA levels and retards mineral deposition relative to vector control. This novel kinase, therefore, is likely to play an important regulatory role in attenuating the program of osteoblast differentiation.Numerous investigations have been directed at elucidating factors that regulate osteoblast differentiation (1). The bone morphogenetic proteins (BMPs) 1 are potent local factors that promote osteoblast differentiation during development as well as during bone remodeling (2). The molecular events downstream of BMP signaling that result in tissue-specific gene expression and skeletal development have only been partially elucidated. The binding of BMPs to their receptors leads to the assembly of a receptor complex in which the type II receptor phosphorylates and activates the type I receptor. As a result, pathway-restricted SMADs are phosphorylated, leading to interactions with the common mediator SMAD, smad4 (3). This complex is then translocated to the nucleus, where it modulates transcription of target genes. BMP signaling can also interfere with the effects of other growth and differentiation factors. It has been demonstrated that BMP-2 treatment of mesangial cells prevents phosphorylation of a transcription factor, Elk1, in response to platelet-derived growth factor (PDGF) signaling. This effectively inhibits PDGF-induced Elk-1-mediated transcription, and blocks PDGF-induced transcription of c-fos, an Elk-1 target (4). BMP-7 has been shown to be a potent inducer of Cbfa1, a transcription factor belonging to the runt-domain gene family that, in turn, regulates the expression of several genes in the osteoblast (5). Although Cbfa1 expression is necessary, it a...

A Novel AP-2 Adaptor Interaction Motif Initially Identified in the Long-splice Isoform of Synaptojanin 1, SJ170

“…The AAK1 693 STWNPFDD sequence is invariant in the presumptive rodent AAK1 orthologues (rat accession number XM_232172 and partial mouse clone AAH43125) and a nearly identical sequence (SGWNPFGE) is also found within the carboxyl-terminal segment of mouse (NM_080708) and human (Q9NSY1) BMP2-inducible protein kinase (BIKe) (48). The kinase domains of AAK1 and BIKe are 77% identical and the rodent and human BIKe sequences each also contain two DPF triplets.…”

Identification of Novel Gene Expression in Healing Fracture Callus Tissue by DNA Microarray

“…Genes involved in inflammation expressed highly within this cluster include IL-2, 4, and 6 signaling including p21, NF-kappa-B inhibitor beta, and complement C3 precursor. Seven of the genes in cluster 3 are involved in bone formation including BMP-2-inducible protein kinase (BIKe) [39] that has been shown to attenuate the program of osteoblast differentiation. This group also contains genes involved in signal transduction, transcription, and cell cycle regulation (including COX-2) [40].…”

Genomic analysis of neural crest induction

“…For example, the secreted factor slit3 is a chemorepellant that binds to robo receptors on axons during axon guidance (reviewed by Guthrie, 2001), although its expression and role in the formation of migratory neural crest cells was previously unrecognized. Meanwhile, BIKe is a serine/threonine kinase upregulated as a downstream response to BMP signaling (Kearns et al, 2001). Confirming the specificity of our screen, we isolated the receptor Notch1, which is expressed in migratory neural crest cells (Wakamatsu et al, 2000) and cell-autonomously required in premigratory neural crest for slug expression (Endo et al, 2002).…”