Yun-Hye Jin

Protein kinase A phosphorylates and regulates dimerization of 14‐3‐3ζ

Recognition of phosphorylated serine/threonine‐containing motifs by 14‐3‐3 depends on the dimerization of 14‐3‐3. However, the molecular cues that control 14‐3‐3 dimerization are not well understood. In order to identify proteins that control 14‐3‐3 dimerization, we analyzed proteins that have effects on 14‐3‐3 dimerization and report that protein kinase A (PKA) phosphorylates 14‐3‐3ζ at a specific residue (Ser58). Phosphorylation by PKA leads to modulation of 14‐3‐3ζ dimerization and affect its interaction with partner proteins. Substitution of Ser58 to Ala completely abolished phosphorylation of 14‐3‐3ζ by PKA. A phospho‐mimic mutant of 14‐3‐3ζ, Ser58 to Glu substitution, failed to form homodimers, showed reduced interaction with 14‐3‐3ε and p53, and could not enhance transcriptional activity of p53. Moreover, activation of PKA decreases and inhibition of PKA increases the dimerization of 14‐3‐3ζ and the functional interaction of 14‐3‐3ζ with p53. Therefore, our results suggest that PKA is a new member of protein kinases that can phosphorylate and impair the function of 14‐3‐3.

Akt phosphorylates and regulates the osteogenic activity of Osterix

Osterix (Osx), a zinc-finger transcription factor is required for osteoblast differentiation and new bone formation during embryonic development. Akt is a member of the serine/threonine-specific protein kinase and plays important roles in osteoblast differentiation. The function of Osterix can be also modulated by post-translational modification. But, the precise molecular signaling mechanisms between Osterix and Akt are not known. In this study, we investigated the potential regulation of Osterix function by Akt in osteoblast differentiation. We found that Akt phosphorylates Osterix and that Akt activation increases protein stability, osteogenic activity and transcriptional activity of Osterix. We also found that BMP-2 increases the protein level of Osterix in an Akt activity-dependent manner. These results suggest that Akt activity enhances the osteogenic function of Osterix, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Osterix, at least in part, through Akt.

Acetylation of histone deacetylase 6 by p300 attenuates its deacetylase activity.

Protein acetyltransferases and deacetylases affect the activities of each other. This is well documented by the acetylation and inhibition of HDAC1 by p300, a transcriptional co-activator with protein acetyltransferase activity. However, the relationship between HDAC6 and p300 is poorly understood. HDAC6 is a class II histone deacetylase and differs from other members of HDAC family in that it contains two HDAC domains and an ubiquitin-binding motif. HDAC6 is a microtubule-associated deacetylase. It predominantly deacetylates non-histone proteins, including alpha-tubulin, and regulates cell motility. Here, we report that p300 interacts with and acetylates HDAC6 resulting down-regulation of HDAC6 deacetylase activity. Furthermore, we provide evidences that acetylation of HDAC6 by p300 inhibits tubulin deacetylation and suppression of Sp1 transcriptional activity by HDAC6. Our results demonstrate that p300 can inactivate HDAC6 by acetylation, and that p300 may regulate the activity of Sp1 indirectly through HDAC6 in addition to its direct modification of Sp1.

Akt enhances Runx2 protein stability by regulating Smurf2 function during osteoblast differentiation

Runx2 plays essential roles in bone formation and chondrocyte maturation. Akt promotes osteoblast differentiation induced by the bone morphogenetic proteins BMP2 and enhances the function and transcriptional activity of Runx2. However, the precise molecular mechanism underlying the relationship between Runx2 and Akt is not well understood. In this study, we examined the role of Akt in regulating Runx2 function. We found that Akt increases the stability of Runx2 protein. However, the level of Runx2 mRNA was not affected by Akt, and we did not find any evidence for direct modification of Runx2 by Akt. Instead, we found evidence that Akt induces the phosphorylation of the Smad ubiquitination regulatory factor Smurf2 and decreases the level of Smurf2 protein through ubiquitin/proteasome‐mediated degradation of Smurf2. Akt also alleviates Smurf2‐mediated suppression of Runx2 transcriptional activity. Taken together, our results suggest that Akt regulates osteoblast differentiation, at least in part, by enhancing the protein stability and transcriptional activity of Runx2 through regulation of ubiquitin/proteasome‐mediated degradation of Smurf2.

Calmodulin-dependent kinase II regulates Dlx5 during osteoblast differentiation.

Calmodulin-dependent kinase II (CaMKII) acts as a key regulator of osteoblast differentiation. CaMKII is a Ca(2+)-activated serine/threonine kinase and it regulates the activity of target proteins by phosphorylation. Dlx5 transcription factor plays crucial roles in osteoblast differentiation. The expression of Dlx5 is regulated by several osteogenic signaling pathways from early stages of osteoblastogenesis. In addition, Dlx5 can be phosphorylated and activated by p38, suggesting that the function of Dlx5 can be also modulated by post-translational modification. Although CaMKII and Dlx5 both play crucial roles during osteoblast differentiation, the interaction between CaMKII and Dlx5 has not been investigated. In the current study, we examined the effects CamKII on the function of Dlx5. We found that CaMKII phosphorylates Dlx5, and that CaMKII increases the protein stability and the osteoblastogenic transactivation activity of Dlx5. Conversely, a CaMKII inhibitor KN-93 decreased the osteogenic and transactivation activities of Dlx5. These results indicate that CaMKII regulates osteoblast differentiation, at least in part, by increasing the protein stability and the transcriptional activity of Dlx5.

ERK1/2 regulates SIRT2 deacetylase activity

SIRT2 is a mammalian member of the Sirtuin family of NAD-dependent protein deacetylases. The function of SIRT2 can be modulated by post-translational modification. However, the precise molecular signaling mechanisms of SIRT2 and extracellular signal-regulated kinase (ERK)1/2 have not been correlated. We investigated the potential regulation of SIRT2 function by ERK1/2. ERK activation by the over-expression of constitutively active MEK increased protein levels and enhanced the stability of SIRT2. In contrast, U0126, an inhibitor of mitogen-activated kinase kinase, suppressed SIRT2 protein level. ERK1/2 interacted with SIRT2 exogenously and endogenously. Deacetylase activity of SIRT2 was up-regulated in an ERK1/2-mediated manner. These results suggest that ERK1/2 regulates SIRT2 by increasing the protein levels, stability and activity of SIRT2.

PKC signaling inhibits osteogenic differentiation through the regulation of Msx2 function.

Protein kinase C (PKC) signaling regulates osteoblast differentiation, but little is known about its downstream effectors. We examined the effect of modulating PKC activity on osteogenic transcription factors and found that the protein level of Msx2 is affected. Msx2 is induced by osteogenic signals such as BMPs and it plays critical roles in bone formation and osteoblast differentiation. Here, we examined the role of PKC signaling in regulating the function of Msx2. We found that the inhibition of PKC signaling enhances osteogenic differentiation in BMP2-stimulated C2C12 cells. Treatment with inhibitors of PKC activity or overexpression of kinase-defective (KD), dominant-negative mutant PKC isoforms strongly reduced the level of Msx2 protein. Several PKC isoforms (α, β, δ, and ζ) interacted with Msx2, and PKCβ phosphorylated Msx2 at Thr135 and Thr141. Msx2 repressed the transcriptional activity of the osteogenic transcription factor Runx2, and this repression was relieved by inhibition of PKC activity or overexpression of the KD mutant PKC isoforms. In addition, PKC prolonged the half-life of Msx2 protein. These results suggest that PKC signaling modulates osteoblast differentiation, at least in part, through the regulation of Msx2.

Yin Yang 1 is a multi-functional regulator of adipocyte differentiation in 3T3-L1 cells.

Yin Yang 1 (YY1) is an ubiquitously distributed transcription factor that belongs to the GLI-Kruppel class of zinc finger proteins. The mechanism by which YY1 regulates adipocyte differentiation remains unclear. In this study, we investigated the functional role of YY1 during adipocyte differentiation. During the early stage, YY1 gene and protein expression was transiently downregulated upon the induction of differentiation, however, it was consistently induced during the later stage. YY1 overexpression decreased adipocyte differentiation and blocked cell differentiation at the preadipocyte stage, while YY1 knockdown by RNA interference increased adipocyte differentiation. YY1 physically interacted with PPARγ (Peroxisome proliferator-activated receptor gamma) and C/EBPβ (CCAAT/enhancer-binding protein beta) respectively in 3T3-L1 cells. Through its interaction with PPARγ, YY1 directly decreased PPARγ transcriptional activity. YY1 ectopic expression prevented C/EBPβ from binding to the PPARγ promoter, resulting in the downregulation of PPARγ transcriptional activity. These results indicate that YY1 repressed adipocyte differentiation by repressing the activity of adipogenic transcriptional factors in 3T3-L1 cells.

Src regulates the activity of SIRT2

SIRT2 is a mammalian member of the Sirtuin family of NAD(+)-dependent protein deacetylases. The tyrosine kinase Src is involved in a variety of cellular signaling pathways, leading to the induction of DNA synthesis, cell proliferation, and cytoskeletal reorganization. The function of SIRT2 is modulated by post-translational modifications; however, the precise molecular signaling mechanism of SIRT2 through interactions with c-Src has not yet been established. In this study, we investigated the potential regulation of SIRT2 function by c-Src. We found that the protein levels of SIRT2 were decreased by c-Src, and subsequently rescued by the addition of a Src specific inhibitor, SU6656, or by siRNA-mediated knockdown of c-Src. The c-Src interacts with and phosphorylates SIRT2 at Tyr104. c-Src also showed the ability to regulate the deacetylation activity of SIRT2. Investigation on the phosphorylation of SIRT2 suggested that this was the method of c-Src-mediated SIRT2 regulation.

Protein kinase A phosphorylates and regulates the osteogenic activity of Dlx5.

Dlx5 transcription factor plays important roles in osteoblast differentiation and its transcription is regulated by many osteogenic signals including BMP-2. Recent studies suggest that the function of Dlx5 is also regulated post-translationally by protein kinases such as p38 and CaMKII. Protein kinase A (PKA) is involved in several steps of osteoblast differentiation and its activity has been shown necessary, yet not sufficient, for BMP-induced osteoblast differentiation. PKA is a ubiquitous cellular kinase that phosphorylates serine and threonine residues(s) of target proteins. In this study, we investigated the potential regulation of Dlx5 function by PKA in osteoblast differentiation. We found that PKA phosphorylates Dlx5 and that PKA activation increases the protein stability, osteogenic activity and transcriptional activity of Dlx5. We also found that BMP-2 increases the protein level of Dlx5 in a PKA activity-dependent manner. These results suggest that PKA activity enhances the osteogenic function of Dlx5, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Dlx5, at least in part, through PKA.