Covalent lipid modifications mediate the membrane attachment and biological activity of Ras proteins. All Ras isoforms are farnesylated and carboxyl-methylated at the terminal cysteine; H-Ras and N-Ras are further modified by palmitoylation. Yeast Ras is palmitoylated by the DHHC cysteine-rich domain-containing protein Erf2 in a complex with Erf4. Here we report that H-and N-Ras are palmitoylated by a human protein palmitoyltransferase encoded by the ZDHHC9 and GCP16 genes. DHHC9 is an integral membrane protein that contains a DHHC cysteine-rich domain. GCP16 encodes a Golgi-localized membrane protein that has limited sequence similarity to yeast Erf4. DHHC9 and GCP16 codistribute in the Golgi apparatus, a location consistent with the site of mammalian Ras palmitoylation in vivo. Like yeast Erf2⅐Erf4, DHHC9 and GCP16 form a protein complex, and DHHC9 requires GCP16 for protein fatty acyltransferase activity and protein stability. Purified DHHC9⅐GCP16 exhibits substrate specificity, palmitoylating H-and N-Ras but not myristoylated G ␣i1 or GAP-43, proteins with N-terminal palmitoylation motifs. Hence, DHHC9⅐GCP16 displays the properties of a functional human ortholog of the yeast Ras palmitoyltransferase.

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Parathyroid hormone (PTH) binds to its receptor PTH1R (parathyroid hormone 1 receptor) in osteoblastic cells to regulate bone remodeling and calcium homeostasis. While prolonged exposure to PTH causes increased bone resorption, intermittent injections of PTH have an anabolic effect on bone. The molecular mechanisms regulating these processes are still largely unknown. Here, we present our results on gene expression profile changes in the PTH-treated osteoblastic cell line, UMR 106-01, using DNA microarray analysis. A total of 125 known genes and 30 unknown expressed sequence tags (ESTs) were found to have at least 2-fold expression changes after PTH treatment at 4, 12, and 24 h. 14 genes were previously known to be PTH-regulated but many were unknown to be regulated by PTH prior to our experiments. Real-time reverse transcriptase-PCR confirmed that 90 and 50% of the genes are regulated more than 2-fold by PTH in UMR 106-01 and rat primary osteoblastic cells, respectively. Most genes belong to the following protein families: hormones, growth factors, and receptors; signal transduction pathway proteins; transcription factors; proteases; metabolic enzymes; structural and matrix proteins; transporters; etc. These results provide a comprehensive and deeper knowledge about PTH regulation of osteoblastic gene expression. Next, we designed a computational method to extract information about transcription factors likely involved in regulating these genes. These factors include those previously known to be involved in PTH signaling (AP-1 and the cAMP response element-binding protein), those that were identified by microarray data (C/EBP), and some novel transcription factors (AP-2, AP-4, SP1, FoxD3, etc.). Our results suggest that a reliable bioinformatics approach can be easily applied for other systems.

Parathyroid hormone (PTH) is known to have both catabolic and anabolic effects on bone. The dual functionality of PTH may stem from its ability to activate two signal transduction mechanisms: adenylate cyclase and phospholipase C. Here, we demonstrate that continuous treatment of UMR 106-01 and primary osteoblasts with PTH peptides, which selectively activate protein kinase C, results in significant increases in DNA synthesis. Given that ERKs are involved in cellular proliferation, we examined the regulation of ERKs in UMR 106-01 and primary rat osteoblasts following PTH treatment. We demonstrate that treatment of osteoblastic cells with very low concentrations of PTH (10 ؊12 to 10 ؊11 M) is sufficient for substantial increases in ERK activity.

Though the mitogenic activity of lysophosphatidic acid (LPA) has been well established through classical studies, its mechanism of action was long obscure. Recent identification and cloning of LPA-specific receptors has led to the elucidation of the G-proteins and signaling pathways through which this molecule functions. In addition to its mitogenic properties, recent reports have suggested that LPA may also promote cell survival. This review will summarize the current literature regarding LPA signaling and its role as an antiapoptotic factor.