1. Volume 8, Issue 5, Pages 959-969 (November 2001)
Unexpected Effects of FERM Domain Mutations on Catalytic Activity of Jak3 
Yong-Jie Zhou, Min Chen, Nancy A. Cusack, Lida H. Kimmel, Kelly S. Magnuson, James G. Boyd, Wen Lin, Joseph L. Roberts, Andrea Lengi, Rebecca H. Buckley, Robert L. Geahlen, Fabio Candotti, Massimo Gadina, Paul S. Changelian, John J. O'Shea 
Molecular Cell 
Volume 8, Issue 5, Pages (November 2001)
DOI: /S (01)

2. Figure 1 Patient-Derived Jak3 FERM Mutations Impair γc Association
(A) Alignment of the FERM domains of Jak3 and ERM proteins. Sequences of the putative subdomain A and A-B linker of human Jak3 were aligned with murine radixin, human moesin, and human ezrin using the MultiAlin program (Corpet, 1988) and the Blosum62 symbol comparison table. Secondary structure elements are indicated above the sequences using the ESPript www interface with radixin as superimposed secondary structure. An asterisk above the sequence alignment indicates mutations in Jak3; purple indicates the SCID patient-derived mutations; and light blue indicates the artificial mutations.
(B) Schematic structure of three FERM lobes (Hamada, et al., 2000) with predicted placement of Jak3 mutations.
(C) Schematic structure of wild-type and mutant Jak3. The SCID patient-derived Jak3 mutants, Del58A, Y100C, and D169E in the FERM domain and C759R in the JH2, and the mutant K855A that has a mutation at the ATP binding-site of Jak3 kinase domain, are illustrated.
(D) FERM mutants inhibit Jak3/γc association. COS-7 cells were transfected with 5 μg of Tac-γc along with the other indicated cDNAs. Cell lysates were immunoprecipitated with anti-Tac mAb and blotted with anti-Jak3 Ab (upper). The membrane was reprobed with anti-γc Ab (middle) and lysates were immunoblotted with anti-Jak3 Ab (bottom) to ascertain equivalent expression of proteins in each condition.
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2 Mutations in Jak3 FERM Domain Disrupt Kinase Activity
(A) Jak3 FERM mutant fails to mediate IL-2-dependent STAT activation. EBV-transformed human B cell lines from a healthy individual (lanes 1 and 2) or a patient homozygous for the Y100C mutation (lanes 3 and 4) were incubated with IL-2 as indicated. Nuclear extracts were then prepared and EMSA was performed by incubating 10 μg of protein with a labeled oligonucleotide derived from the GAS-like element in the CD23 promoter.
(B) The patient-derived FERM mutation abrogates Jak3 kinase activity. In vitro kinase assays of Jak3 were performed on immune complexes from a normal individual (lane 1) and the patient with the Y100C mutation (lane 2) at room temperature for 5 min. Samples were then separated by SDS-PAGE, transferred to nitrocellulose, and subjected to autoradiography (upper) and blotting with anti-Jak3 Ab (bottom).
(C and D) Patient-derived FERM domain mutations abrogate kinase activity. COS-7 cells were transfected with 5 μg of the indicated cDNAs. The cells were lysed and immunoprecipitated with anti-Jak3 Ab. In vitro kinase activity of Jak3 was measured in the presence of an exogenous substrate, GST-γc at room temperature for 5 and 10 min, respectively. Samples were then separated by SDS-PAGE, transferred to nitrocellulose, and subjected to autoradiography. Autophosphorylation of Jak3 (C) and phosphorylation of GST-γc (D) are shown in the upper panels. Membranes were reprobed with anti-Jak3 Ab (C) and anti-GST Ab (D) to verify equal loading (bottom).
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3
Multiple FERM Mutations Inhibit Jak3 Catalytic Activity and γc Association
Mutations of conserved residues in the Jak3 FERM domain disrupt catalytic activity (A and C) and receptor binding (B). (D) The Jak3 N terminus is required for catalytic activity. COS-7 cells were transfected with 5 μg of the indicated cDNAs. Cell lysates were immunoprecipitated with anti-Jak3 Ab, and in vitro kinase activity was assessed at room temperature for 10 min ([A], [C], [D], and top). Membranes were probed with anti-Jak3 antibody for equal loading (bottom). (B) Cell lysates were immunoprecipitated with anti-Tac mAb and blotted with anti-Jak3 Ab (upper), followed by reblotting with anti-γc Ab (middle). Expression levels of various Jak3 proteins were detected by immunoblotting with anti-Jak3 Ab (bottom).
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4 FERM Mutants Do Not Bind an ATP Analog, FSBA
Transfected COS-7 cells were lysed and immunoprecipitated with anti-Jak3 Ab. (A and B) 14C-FSBA binds specifically to Jak3. (C and D) Catalytically inactive Jak3 mutants are unable to bind 14C-FSBA. The immune complexes from cells transfected with wild-type and mutant Jak3 were incubated with 14C-FSBA at 30°C for 60 min using the conditions for a standard in vitro kinase assay with or without excess unlabeled ATP. Samples were then washed and subjected to SDS-PAGE and autoradiography ([A], [C], and upper). The radioactivity of each labeled protein was quantitated by Phosphoimager analysis and was depicted as counts per minute of 14C-FSBA labeled protein (B and D). The level of expression in each sample was determined by blotting with anti-Jak3 Ab ([A] and [C] bottom panel).
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 5
The Jak3 FERM Domain Binds the Kinase Domain and Can Regulate Its Activity
(A and D) Jak3 FERM domain associates with the JH1 domain. COS cells were transfected with Flag-JH1 (1 μg, [A]), together with 1 μg of each Jak3 C-terminal deletion mutant [lanes 1–3: J3(ΔJH1): FERM-SH2-JH2-aa 1–813; J3(NcoI): FERM-SH2-aa 1–530; and J3(StuI): FERM-aa 1–192] and Tac-γc (lane 4) or transfected with Flag-JH1 (1 μg, [D]), together with 1 μg of J3(StuI) (lane 1), the J3(StuI) cDNA containing Y100C mutation and Tac-γc (lane 3), respectively. Lysates were immunoprecipitated with a Jak3 N-terminal anti-peptide Ab (lanes 1–3 in [A] and lanes 1 and 2 in [D]) or anti-Tac-mAb (lane 4 in [A] and lane 3 in [D]), and blotted with anti-Flag mAb (upper panel). Expression of the Jak3 deletion mutants (second panel), Tac-γc (third panel), and Flag-JH1 (bottom panel) was assessed by blotting with indicated antibodies.
(B, C, and E) Overexpression of the FERM domain modulates kinase activity. COS cells were transfected with the kinase domain construct, Flag-JH1 (0.5 μg) in the absence (lanes 1 and 2 in [B] and lane 1 in [C] and [E]) or presence of the indicated amounts of the FERM domain constructs, J3(StuI) (lanes 3–8 in [B], lanes 2–4 in [C] and lanes 2–3 in [E]) or J3(StuI/Y100C) (lanes 4 and 5 in [E]). Lysates were immunoprecipitated with anti-Flag mAb and then subjected to an in vitro kinase assay ([B], upper; [C and E], inserts). The level of expression was determined by blotting with indicated Ab ([B], middle and bottom). Incorporated radioactive phosphate was quantitated by Phosphoimager analysis and is expressed as counts per minute of 32P-labeled protein (C and E).
Molecular Cell 2001 8, DOI: ( /S (01) )

7. Figure 6 Staurosporine Binding Disrupts Jak3/γc Association
(A) Recombinant Jak3 and γc associate in vitro. ELISA plates were coated with GST (open squares) or GST-γc (closed diamonds) proteins and then incubated with purified Jak3 protein for various times. After washing, anti-Jak3 Ab binding was used to measure Jak3/γc association.
(B) Inhibition of Jak3/γc binding by a γc peptide. The recombinant Jak3/γc proteins were incubated as described in (A); but prior to the addition of anti-Jak3 Ab, a wild-type γc-peptide (open diamonds) or a SCID patient-derived γc-peptide (open squares) was added to disrupt the association. As a control, GST and Jak3 incubated with a wild-type γc peptide is shown (closed squares). After washing, we used anti-Jak3 Ab to detect the Jak3/γc association.
(C) A Jak3 mutant lacking the kinase and pseudokinase domains binds to γc.
(D) Staurosporine inhibits binding of the full-length Jak3 to γc, but has no effect on a Jak3 mutant lacking the kinase and pseudokinase domains. The recombinant Jak3 and GST-γc proteins were incubated as described in (A); but prior to the addition of anti-Jak3 Ab, varying concentrations of staurosporine were added.
(E) Staurosporine inhibition of Jak3/γc association is competed by ATP. Jak3 and GST-γc were incubated without (open diamonds) or with μM (closed diamonds), 0.15 μM (open squares), or 0.3 μM staurosporine (SP, closed squares) in the presence of indicated concentrations of ATP.
Molecular Cell 2001 8, DOI: ( /S (01) )

8. Figure 7
Staurosporine Inhibits Association of Prebound Jak3/γc Proteins from Cells
COS-7 cells were cotransfected with the indicated cDNAs and were lysed 36 hr after transfection.
(A) Cell lysates were immunoprecipitated with anti-Tac mAb, and Jak3/γc complexes were incubated without (lanes 1 and 6) or with staurosporine (lanes 2–5) or the Src kinase inhibitor PP2 (lanes 7–10) at indicated concentrations at room temperature for 1 hr. The immune complexes were then washed, subjected to SDS-PAGE, transferred to nitrocellulose, and detected by blotting with anti-Jak3 Ab (upper). The same membrane was reprobed with anti-γc Ab (middle). Cell lysates containing Jak3 and each mutant were also detected by blotting with anti-Jak3 Ab (bottom).
(B) The cell lysates were immunoprecipitated with anti-Jak3 Ab, and in vitro kinase activity was performed in the absence (lanes 1 and 6) or in the presence of various concentrations of staurosporine (lanes 2–5) or PP2 (lanes 7–10). Samples were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to autoradiography (upper). Membranes were probed with anti-Jak3 Ab to ascertain equal loading (bottom).
Molecular Cell 2001 8, DOI: ( /S (01) )

9. Figure 8
A Model for Interactions of the Jak3 FERM Domain with the Kinase Domain and γc
Molecular Cell 2001 8, DOI: ( /S (01) )