Src-family kinases __________________________________________________________________

Src was the first proto-oncogenic protein described. Since then,  many other tyrosine kinases have been identified and characterized. Src remains the ‘grandfather’ non-receptor tyrosine kinase and serves as a prototype for understanding the function and regulation of other tyrosine kinases (refs)

The Src family is composed of nine members in vertebrates:

Src, Yes, Fgr, Yrk, Fyn, Lyn, Hck, Lck and Blk click image on right for a family tree based on sequence similarity

Several members of the family are found in all tissues while others are more specialized (refs).

Members of the Src-family share a common structure.  

Coloring in this table corresponds to the parts of Src-family proteins.  Click the image above for a larger view of the general layout of Src-family members.

The three dimensional structure of the Src-family of proteins was revealed in 1997 when the crystal structures of inactive human Src, human Hck and chicken Src were solved (refs). These structures lack the N-terminal unique domain.

Click the images below for a larger version (ref)

Src

Hck

There is also a structure for an active form of Src

The original model of activation is based on comparison of inactive Src with the catalytic domain of an active Src-family member (Lck) (refs).

Click image for a larger version

As shown in the figure above, the activation loop of Lck is swung out, relative to the activation loop of Src loop, and a tyrosine in the activation loop (Tyr416 in Src) is phosphorylated. In addition, the N-lobe and particularly the C-helix within the N-lobe of the catalytic domain undergo conformational changes upon activation. The lobes also rotate with respect to each other.

In 2005, several structure of active Src kianse domains were revealed (1YOJ-1YOM) (refs).  In addition, a crystal of an active form was also determined that contained SH3, SH2 and the kinase domain (1Y57).

Click here to see an animation that compares the conformations of an inactive Src-family member to an active member in the regions surrounding the activation loop. Destabilization of the activation loop and rotation of the C-helix leads to repositioning of a glutamic acid (Glu310) from the C-helix, such that it ion-pairs with Lys295 in the active site. This interaction positions Lys295 for proper interaction with ATP to permit nucleophilic attack (refs).

The human immunodeficiency virus (HIV) expresses a number of unique proteins.  Among these is the Nef protein.  Nef is responsible for downregulation of CD4 receptors and for enhacement of viral replication.  Humans infected with a mutant form of HIV in which Nef is deleted or mutated do not develop AIDS (refs).

One of the proteins that Nef interacts with is hematopoietic cell kinase (Hck).  The binding constant for HIV Nef and Hck SH3 is the tightest binding for any known association of an SH3 domain (Kd=0.25uM) (refs).

Please visit these links for more info about Nef

There are two x-ray crystal structures of HIV Nef and one NMR solution structure.  The images on the left were taken from John Kuriyan's web page.  HIV Nef interacts with Hck SH3 via a proline-rich 12 amino acid region.  There is additional interaction with amino acids in the RT-loop of SH3.  The interactions can be seen in the top figure where Fyn SH3 is in blue and sit atop the core of HIV Nef.  the lower figure shows a space-filling model of Nef and highlights the the fact that Nef binds to Fyn SH3 not only via the conventional proline-rich ligand, but also has additional binding / speceficity regions (refs). Please visit these links for more information PDB entries    2NEF - NMR solution structure    1EFN - complex of Fyn SH3 and Nef    1BU1 - complex of Hck SH3 and Nef

The binding of HIV Nef and Hck/Fyn SH3 appears to be responsible for increasing the replicative ability of HIV.  CD4 downregulation is not modulated by interactions with Hck or other Src-family kinases (refs).

Src-family

Superti-Furga, G. & Courtneidge, S. (1995) Regulation of the Src protein tyrosine kinase. BioEssays. 17(4), 321-330.

Brown, M.T. and Cooper, J.A. (1996) Regulation, substrates and functions of Src. Biochim. Biophys. Acta. 1287, 121–149.

Thomas, S.M. and Brugge, J.S. (1997) Cellular functions regulated by Src family kinases. Annu. Rev. Cell Dev. Biol. 13, 513–609.

Lowell CA and Soriano P (1996) Knockouts of Src-family kinases: stiff bones, wimpy T cells and bad memories. Genes Dev. 10, 1845–1857.

Schlessinger J (2000) New roles for Src kinases in control of cell survival and angiogenesis. Cell 100, 293–296.

Xu, W., Harrison, S.C. and Eck , M.J. (1997) Three-dimensional structure of the tyrosine kinase c-Src. Nature 385, 595–602.

Sicheri, F., Moarefi, I. and Kuriyan, J. (1997) Crystal structure of the Src family tyrosine kinase Hck. Nature 385, 602–609.

Williams J.C. et al. (1997). The 2.35 A crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regultory interactions. J. Mol. Biol. 274 (5) 757-775.

Williams, J.C., Wierenga, R.K. and Saraste M (1998) Insights into Src kinase functions: structural comparisons. Trends Biochem. Sci. 23, 179–184.

Hubbard, S.R. (1999) Src autoinhibition: let us count the ways. Nat. Struct. Biol. 6, 711–714.

Gonfloni, S., Weijland, A., Kretzschmar, J. and Superti-Furga, G. (2000) Crosstalk between the catalytic and regulatory domains allows bidirectional regulation of Src. Nat Struct Biol. 7(4), 281–286.

Cowan-Jacob, S.W., Fendrich, G., Manley, P.W., Jahnke, W., Fabbro, D., Liebetanz, J., and Meyer T. (2005). The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation. Structure 13(6):861-71.

Breitenlechner, C.B., Kairies, N.A., Honold, K., Scheiblich, S., Koll, H., Greiter, E., Koch, S., Schafer, W., Huber, R., Engh, R.A. (2005).  Crystal structures of active SRC kinase domain complexes. J Mol Biol. 353(2):222-31.
 

HIV Nef

Peter, F. (1998). HIV Nef: the mother of all evil. Immunity 9: 433-437.

Cullen, B. R. (1999). HIV-1 Nef protein: an invitation to a kill. Nat. Medicine 5(9): 985-986.

Stevenson, M. (1996). Pathway to understanding AIDS. Nat. Struct. Biol 3(4): 303-6

Grzesiek, S., A. Bax, et al. (1996). The solution structure of HIV-1 Nef reveals an unexpected fold and permits delineation of the binding surface for the SH3 domain of Hck tyrosine protein kinase. Nat. Struct. Biol 3(4): 340-5.

Lee, C.-H., B. Leung, et al. (1995). A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein. EMBO J. 14(20): 5006-15.

Lee, C.-H., K. Saksela, et al. (1996). Crystal structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 domain. Cell 85(6): 931-942.

Arold, S., R. O'Brien, et al. (1998). RT loop flexibility enhances the specificity of Src family SH3 domains for HIV-1 Nef. Biochemistry 37(42): 14683-91.