Nuclear factor B (NF-B)-inducing kinase (NIK),The IGFs 1 are the only known growth factors that are crucial to myogenesis (1). IGF-I and IGF-II switch on the myogenic program through the IGF-I receptor (2), activating the expression of myogenic transcription factors, cell cycle arrest, musclespecific protein expression, and cell fusion to form multinucleated myotubes (3, 4). PI3K is an essential second messenger for myogenesis (5-9). We have recently described a myogenic signaling cascade initiated by IGF-II that leads to biochemical and morphological skeletal muscle cell differentiation and that involves PI3K activation, NF-B activation, and inducible nitricoxide synthase expression and activation (10). In this report, we further analyze the role of the NF-B-activating signaling cascade in myogenesis. NF-B transcription factors are key mediators of inflammatory responses, immune system functioning, transformation, oncogenesis, and anti-apoptotic signaling (11-13). NF-B exists in the cytoplasm in an inactive form by virtue of its association with inhibitory proteins termed IB (11-15). NF-B translocation to the nucleus and activation are most frequently achieved through the signal-induced proteolytic degradation of IB in the cytoplasm. Two kinases, IKK␣ and IKK, which are contained in a high-molecularweight multiprotein complex, show inducible IB kinase activity and play a key role in NF-B activation by a variety of stimuli (16 -19). Despite their high sequence similarity, IKK␣ and IKK have different regulatory and functional roles. In mice lacking IKK, the activation of NF-B by cytokines is abolished, and mouse embryos die on days 12-13 of gestation due to massive liver apoptosis (20). In contrast, IKK␣ is dispensable for pro-inflammatory responses, but plays an essential role in embryonic development. Mice lacking IKK␣ exhibit defective proliferation and differentiation of epidermal keratinocytes and defective limb and skeletal patterning (21,22). IKK␣ and IKK are themselves phosphorylated and activated by one or more upstream kinases, like NIK, which is a member of the mitogen-activating protein kinase kinase kinase family (23-25).We report here that IB␣ phosphorylation at Ser-32 and Ser-36 is required for both IGF-II-dependent NF-B activation and differentiation in L6E9 myoblasts. We show that IKK␣ is involved in IGF-II-dependent multinucleated myotube formation and muscle-specific gene expression, whereas IKK is not essential for these processes. Our data suggest that NIK activation triggers myogenin expression and multinucleated myotube formation in the absence of IGF-II.