Increased osteoblastic c-fos expression by parathyroid hormone requires protein kinase A phosphorylation of the cyclic adenosine 3′,5′-monophosphate response element-binding protein at serine 133

PTH induces c-fos expression rapidly and transiently in osteoblastic cells and requires the activity of the cAMP response element-binding protein (CREB). Here we provide evidence that protein kinase A (PKA) is the enzyme responsible for phosphorylating CREB at serine 135 (S133) and that this event is required for PTH-induced c-fos expression. PTH increases the level of phosphorylation of CREB at S133 in a time- and dose-dependent manner, correlating with the time and level of activation of PKA in response to PTH. PTH-(1-34) and -(1-31), each known to activate the cAMP pathway, induced the phosphorylation of CREB and increased the levels of c-fos messenger RNA, whereas PTH-(3-34), -(13-34), and -(28-48) could not. Specific inhibitors of calcium/calmodulin-dependent protein kinases and protein kinase C could not inhibit CREB phosphorylation or c-fos expression in response to PTH; however, H-89, a specific inhibitor of PKA, could do so in a dose-dependent manner. In addition, PTH-induced c-fos promoter activity was completely inhibited in a dose-dependent fashion by transfection of the heat-stable inhibitor of PKA. Taken together, these data provide strong evidence that PKA is the enzyme responsible for phosphorylating CREB at S133 in response to PTH and that PKA activity is required for PTR-induced c-fos expression.