Activation of either protein kinase C␣ (PKC␣) or c-Src results in similar effects on cell morphology, including changes in actin filament integrity, cell shape changes, and stimulation of signals associated with increased motility and invasion (20,21,23). Several studies support the existence of cross talk between c-Src-and PKC␣-mediated signaling pathways. Constitutive activation of c-Src or stable expression of v-Src will concomitantly stimulate an increase in PKC␣ signaling (8,37,44). These data indicate that PKC␣ could function downstream of c-Src. However, other data support a hypothesis that PKC␣ can signal upstream of c-Src and stimulate c-Src activity. Activation of PKC␣ has been demonstrated to initiate changes in actin filaments that resemble those that occur in Src 527F -transformed cells (7,9,11,16). Likewise, it was demonstrated that stimulation of cells with phorbol esters will direct activation of c-Src in SH-SY5Y cells (5), while treatment of mouse epidermis with the tumor promoter tetradecanoyl phorbol acetate induced dose-dependent increases in c-Src kinase activity and the phosphotyrosine content of the ErbB2 receptor, which correlated with tumor-promoting ability (43). Furthermore, it was demonstrated that PKC␣ can directly stimulate c-Src activity in A7r5 rat aortic smooth muscle cells, and the induction of Src kinase activity is necessary for PKC-mediated actin reorganization (3). Thus, these data indicate that PKC␣ may function upstream as an activator of c-Src. Although PKC␣ is able to phosphorylate c-Src (15, 31), in vitro studies indicate that PKC␣ does not activate c-Src directly (4, 29). Thus, although these reports demonstrate the ability of PKC␣ to direct activation of c-Src, the mechanism of PKC␣-mediated c-Src activation is unclear.AFAP-110 is an adaptor protein that has been demonstrated to bind to Src via SH2 and SH3 interactions (17,18) and that will bind to PKC␣ via the amino-terminal pleckstrin homology (PH1) domain (32). A carboxy-terminal leucine zipper (Lzip) motif stabilizes AFAP-110 multimer formation and provides an autoinhibitory regulatory function for 32,33,35). While expression of wild-type AFAP-110 has little effect on cell morphology, deletion of the Lzip motif (AFAP-110 ⌬Lzip ) followed by ectopic expression of AFAP-110 ⌬Lzip

The actin ®lament-associated protein of 110 kDa (AFAP-110) is a Src binding partner that represents a potential modulator of actin ®lament integrity in response to cellular signals. Previous reports have demonstrated that AFAP-110 is capable of directly binding and altering actin ®laments. Deletion of the leucine zipper motif of AFAP-110 (AFAP-110 Dlzip ) has been shown to induce a phenotype which resembles Srctransformed cells, by repositioning actin ®laments into rosettes. This deletion also mimics a conformational change in AFAP-110 that is detected in Src-transformed cells. The results presented here indicate that unlike AFAP-110, AFAP-110 Dlzip is capable of activating cellular tyrosine kinases, including Src family members, and that AFAP-110 Dlzip itself is hyperphosphorylated. The newly tyrosine phosphorylated proteins and activated Src-family members appear to be associated with actinrich lamellipodia. A point mutation that alters the SH3-binding motif of AFAP-110 Dlzip prevents it from activating tyrosine kinases and altering actin ®lament integrity. In addition, a deletion within a pleckstrin homology (PH) domain of AFAP-110 Dlzip will also revert its e ects upon actin ®laments. Lastly, dominant-positive RhoA V14 will block the ability of AFAP-110 Dlzip from inducing actin ®lament rosettes, but does not inhibit Src activation. Thus, conformational changes in AFAP-110 enable it to activate cellular kinases in a mechanism requiring SH3 and/or PH domain interactions. We hypothesize that cellular signals which alter AFAP-110 conformation, enable it to activate cellular kinases such as cSrc, which then direct changes in actin ®lament integrity in a Rho-dependent fashion. Oncogene (2001) 20, 6607 ± 6616.

AFAP-110 has an intrinsic ability to alter actin filament integrity as an actin filament crosslinking protein. This capability is regulated by a carboxy terminal leucine zipper (Lzip) motif. The Lzip motif facilitates self-association stabilizing the AFAP-110 multimers. Deletion of the Lzip motif (AFAP-110(Deltalzip)) reduces the stability of the AFAP-110 multimer and concomitantly increases its ability to crosslink actin filaments, in vitro, and to activate cSrc and alter actin filament integrity, in vivo. We sought to determine how the Lzip motif regulates AFAP-110 function. Substitution of the c-Fos Lzip motif in place of the AFAP-110 Lzip motif (AFAP-110(fos)) was predicted to preserve the alpha-helical structure while changing the sequence. To alter the structure of the alpha-helix, a leucine to proline mutation was generated in the AFAP-110 alpha-helical Lzip motif (AFAP-110(581P)), which largely preserved the sequence. The helix mutants, AFAP-110(Deltalzip), AFAP-110(fos), and AFAP-110(581P), demonstrated reduced multimer stability with an increased capacity to crosslink actin filaments, in vitro, relative to AFAP-110. An analysis of opposing binding sites indicated that the carboxy terminus/Lzip motif can contact sequences within the amino terminal pleckstrin homology (PH1) domain indicating an auto-inhibitory mechanism for regulating multimer stability and actin filament crosslinking. In vivo, only AFAP-110(Deltalzip) and AFAP-110(581P) were to activate cSrc and to alter cellular actin filament integrity. These data indicate that the intrinsic ability of AFAP-110 to crosslink actin filaments is dependent upon both the sequence and structure of the Lzip motif, while the ability of the Lzip motif to regulate AFAP-110-directed activation of cSrc and changes in actin filament integrity in vivo is dependent upon the structure or presence of the Lzip motif. We hypothesize that the intrinsic ability of AFAP-110 to crosslink actin filaments or activate cSrc are distinct functions.

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