Induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA in mouse skin organ culture was blocked by two pan-ErbB receptor tyrosine kinase (RTK) inhibitors but not by genetic ablation of ErbB1, suggesting involvement of multiple ErbB species in skin physiology. Human skin, cultured normal keratinocytes, and A431 skin carcinoma cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4. Skin and A431 cells expressed more ErbB3 than did keratinocytes. Despite strong expression of ErbB2 and ErbB3, heregulin was inactive in stimulating tyrosine phosphorylation in A431 cells. In contrast, it was highly active in MDA-MB-453 breast carcinoma cells. ErbB2 displayed punctate cytoplasmic staining in A431 and keratinocytes, compared to strong cell surface staining in MDA-MB-453. In skin, ErbB2 was cytoplasmic in basal keratinocytes, assuming a cell surface pattern in the upper suprabasal layers. In contrast, ErbB1 retained a cell surface distribution in all epidermal layers. Keratinocyte proliferation in culture was found to be ErbB1-RTK-dependent, using a selective inhibitor. These results suggest that in skin keratinocytes, ErbB2 transduces ligand-dependent differentiation signals, whereas ErbB1 transduces ligand-dependent proliferation/survival signals. Intracellular sequestration of ErbB2 may contribute to the malignant phenotype of A431 cells, by allowing them to respond to ErbB1-dependent growth/survival signals, while evading ErbB2-dependent differentiation signals.

We previously provided evidence that the protein encoded by the highly conserved skb1 gene is a putative regulator of Shk1, a p21Cdc42͞Rac -activated kinase (PAK) homolog in the fission yeast Schizosaccharomyces pombe. skb1 null mutants are viable and competent for mating but less elongate than wild-type S. pombe cells, whereas cells that overexpress skb1 are hyperelongated. These phenotypes suggest a possible role for Skb1 as a mitotic inhibitor. Here we show genetic interactions of both skb1 and shk1 with genes encoding key mitotic regulators in S. pombe. Our results indicate that Skb1 negatively regulates mitosis by a mechanism that is independent of the Cdc2-activating phosphatase Cdc25 but that is at least partially dependent on Shk1 and the Cdc2 inhibitory kinase Wee1. We provide biochemical evidence for association of Skb1 and Shk1 with Cdc2 in S. pombe, suggesting that Skb1 and Shk1 inhibit mitosis through interaction with the Cdc2 complex, rather than by an indirect mechanism. These results provide evidence of a previously undescribed role for PAK-related protein kinases as mitotic inhibitors. We also describe the cloning of a human homolog of skb1, SKB1Hs, and show that it can functionally replace skb1 in S. pombe. Thus, the molecular functions of Skb1-related proteins have likely been substantially conserved through evolution.

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In this study , we examined ErbB1 signaling in human basal and squamous cell carcinomas (BCC and SCC) of the skin in vivo. We used enzyme-linked immunosorbent assay , laser capture microdissection-coupled real-time reverse transcriptase-polymerase chain reaction , and immunohistochemistry to assess expression and activation levels of ErbB1 protein , ligands , and potential downstream effectors , in BCC and SCC tumors , stroma , and adjacent epidermis. Although total ErbB1 protein and mRNA were similar in cancerous and normal skin , we found that ErbB1 activation (phospho-Tyr 1068 ) was greater in bulk SCC versus BCC or normal skin. In addition , three ErbB1 ligand transcripts (amphiregulin , heparin-binding epidermal growth factorlike growth factor , and transforming growth factor-␣) were up-regulated in tumor cells of SCC but not BCC. Expression of these ligands was also increased in asymptomatic epidermis adjacent to both SCC and BCC , relative to normal skin. Interestingly , betacellulin transcript levels were inversely regulated compared with the other ligands. Consistently , downstream ErbB1 effectors (Erk1/2 and Akt) were activated in tumor cells of SCC but not of BCC and in adjacent epidermis of both BCC and SCC. These results demonstrate that ErbB1 signaling is hyperactive in tumor cells of SCC but not of BCC and in nearby asymptomatic epidermis of both tumor types. Our results suggest that targeting ErbB1 signaling might be of benefit in the treatment of

ErbB signaling through extracellular signal-regulated kinase (ERK) has been implicated in regulating the expression of ErbB ligands in hyperproliferative skin disorders and wound healing. Here, we characterize the process of autocrine ERK activation in cultured normal human keratinocytes (NHKs) subjected to growth factor (GF) deprivation. Basal ERK phosphorylation was lower after 48 h than after 24 h of GF deprivation, and lowest at 30 -60 min after an additional medium change. ERK phosphorylation was markedly increased by low concentrations of epidermal growth factor (EGF) (0.2-1 ng/ml) that provoked only a limited increase in ErbB1 tyrosine phosphorylation and internalization. Basal ErbB tyrosine phosphorylation and ERK phosphorylation were inhibited by two different ErbB receptor tyrosine kinase inhibitors, by the ErbB1-specific neutralizing monoclonal antibody 225 IgG, by two different metalloproteinase inhibitors, and by neutralizing antibodies against amphiregulin (AR). In contrast, these responses were unaffected by neutralizing antibodies against other ErbB1 ligands or the ErbB2 inhibitors geldanamycin and AG825. The time course of autocrine ERK phosphorylation correlated with the appearance of soluble AR, and two different metalloproteinase inhibitors blocked AR release. These results define an amphiregulin-and ErbB1-dependent mechanism by which autocrine ERK activation is maintained in NHKs, even when ErbB1 autophosphorylation and internalization are limited. INTRODUCTIONThe mammalian c-ErbB family is comprised of four closely related receptor tyrosine kinases (RTKs) that interact hierarchically in response to multiple ErbB receptor ligands (Klapper et al., 2000;Olayioye et al., 2000). Ligand binding to the extracellular domain promotes receptor homo-and heterodimerization, resulting in phosphorylation of specific tyrosine residues on the cytoplasmic domain. These events lead to activation of multiple signal transduction pathways via Src homology 2 (SH2)-and phosphotyrosine binding (PTB)-domain containing cytoplasmic proteins, ultimately affecting many cellular functions, including cell migration, proliferation, and differentiation (Hubbard et al., 1998;Hackel et al., 1999). We and others have demonstrated that human skin expresses ErbB1, ErbB2, and ErbB3, but little or no ErbB4 (Press et al., 1990;Prigent et al., 1992;De Potter et al., 2001;Stoll et al., 2001).ErbB signaling plays a very important role in the reepithelialization of skin wounds, as evidenced by acceleration of burn or partial-thickness wound healing by epidermal growth factor (EGF) (Brown et al., 1989), transforming growth factor-␣ (TGF-␣) (Schultz et al., 1987), heparin-binding EGF-like growth factor (HB-EGF) (Cribbs et al., 1998), and epiregulin (Draper et al., 2003). Corneal wound healing also is markedly inhibited after treatment with ErbB receptor tyrosine kinase inhibitors (RTKIs) (Nakamura et al., 2001). Organ cultures of skin display many features of early wounds, including rapid keratinocyte cytoskeletal alterations, an early ...