Identification of the major sites of autophosphorylation of the murine protein-tyrosine kinase Syk.
The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.
Furlong
M T
MT
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
Mahrenholz
A M
AM
Kim
K H
KH
Ashendel
C L
CL
Harrison
M L
ML
Geahlen
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000737:chemistry*; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D051966:Phospholipase C gamma; D019000:Phosphotyrosine / Q000737:chemistry*; D011494:Protein Kinases / Q000737:chemistry; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D000072377:Syk Kinase; D010738:Type C Phospholipases / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*; D018909:src Homology Domains
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases; C091973:autophosphorylation-dependent multifunctional protein kinase; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D010738:Type C Phospholipases; D051966:Phospholipase C gamma