Characterization of regulatory events associated with membrane targeting of p90 ribosomal S6 kinase 1

RSK is a serine/threonine kinase containing two distinct catalytic domains. Found at the terminus of the Ras/extracellular signal-regulated kinase (ER K)-mitogen-activated protein kinase (MAPK) kinase cascade, mitogen-stimulat ed ribosomal S6 kinase (RSK) activity requires multiple inputs. These input s include phosphorylation of the C-terminal kinase domain activation loop b y ERK1/2 and phosphorylation of the N-terminal kinase domain activation loo p by phosphoinositide-dependent protein kinase-1 (PDK1). Previous work has shown that upon mitogen stimulation, RSK accumulates in the nucleus. Here w e show that prior to nuclear translocation, epidermal growth factor-stimula ted RSK1 transiently associates with the plasma membrane. Myristylation of wild-type RSK1 results in an activated enzyme in the absence of added growt h factors. When RSK is truncated at the C terminus, the characterized ERK d ocking is removed and RSK phosphotransferase activity is completely abolish ed. When myristylated, however, this myristylated C-terminal truncated form (myrCTT) is activated at a level equivalent to myristylated wild-type (myr WT) RSK. Both myrWT RSK and myrCTT RSK can signal to the RSK substrate e-Fo s in the absence of mitogen activation. Unlike myrWT RSK, myrCTT RSK is not further activated by serum. Only the myristylated RSK proteins are basally phosphorylated on avian RSK1 serine 381, a site critical for RSK activity. The myristylated and unmyristylated RSK constructs interact with PDKI upon mitogen stimulation, and this interaction is insensitive to the MEK inhibi tor UO126. Because a kinase-inactive CTT RSK can be constitutively activate d by targeting to the membrane, we propose that ERK may have a dual role in early RSK activation events: preliminary phosphorylation of RSK and escort ing RSK to a membrane-associated complex, where additional MEK/ERK-independ ent activating inputs are encountered.