INHIBITION OF A SIGNALING PATHWAY IN CARDIAC-MUSCLE-CELLS BY ACTIVE MITOGEN-ACTIVATED PROTEIN-KINASE KINASE

Signaling via the Pas pathway involves sequential activation of Ras, R af-1, mitogen-activated protein kinase kinase (MKK), and the extracell ular signal-regulated (ERK) group of mitogen-activated protein (MAP) k inases. Expression from the c-Fos, atrial natriuretic factor (ANF), an d myosin light chain-2 (MLC-2) promoters during phenylephrine-induced cardiac muscle cell hypertrophy requires activation of this pathway. F urthermore, constitutively active Ras or Raf-1 can mimic the action of phenylephrine in inducing expression from these promoters. In this st udy, we tested whether constitutively active MKK, the molecule immedia tely downstream of Raf, was sufficient to induce expression. Expressio n of constitutively active MKK induced ERK2 kinase activity and caused expression from the c-Fos promoter, but did not significantly activat e expression of reporter genes under the control of either the ANF or MLC-2 promoters. Expression of CL100, a phosphatase that inactivates E RKs, prevented expression from all of the promoters. Taken together, t hese data suggest that ERK activation is required for expression from the Fos, ANF, and MLC-2 promoters but MKK and ERK activation is suffic ient for expression only from the Fos promoter. Constitutively active MKK synergized with phenylephrine to increase expression from a c-Fos- or an AP1-driven reporter. However, active MKK inhibited phenylephrin e- and Raf-1-induced expression from the ANF and MLC-2 promoters. A DN A sequence in the MLC-2 promoter that is a target for inhibition by ac tive MKK, but not CL100, was mapped to a previously characterized DNA element (HF1) that is responsible for cardiac specificity. Thus, activ ation of cardiac gene expression during phenylephrine-induced hypertro phy requires ERK activation but constitutive activation by MKK can inh ibit expression by targeting a DNA element that controls the cardiac s pecificity of gene expression.